Glycometabolism and lipid metabolism related genes predict the prognosis of endometrial carcinoma and their effects on tumor cells.

BACKGROUND: Glycometabolism and lipid metabolism are critical in cancer metabolic reprogramming. The primary aim of this study was to develop a prognostic model incorporating glycometabolism and lipid metabolism-related genes (GLRGs) for accurate prognosis assessment in patients with endometrial carcinoma (EC). METHODS: Data on gene expression and clinical details were obtained from publicly accessible databases. GLRGs were obtained from the Genecards database. Through nonnegative matrix factorization (NMF) clustering, molecular groupings with various GLRG expression patterns were identified. LASSO Cox regression analysis was employed to create a prognostic model. Use rich algorithms such as GSEA, GSVA, xCELL ssGSEA, EPIC,CIBERSORT, MCPcounter, ESTIMATE, TIMER, TIDE, and Oncoppredict to analyze functional pathway characteristics of the forecast signal, immune status, anti-tumor therapy, etc. The expression was assessed using Western blot and quantitative real-time PCR techniques. A total of 113 algorithm combinations were combined to screen out the most significant GLRGs in the signature for in vitro experimental verification, such as colony formation, EdU cell proliferation, wound healing, apoptosis, and Transwell assays. RESULTS: A total of 714 GLRGs were found, and 227 of them were identified as prognostic-related genes. And ten GLRGs (AUP1, ESR1, ERLIN2, ASS1, OGDH, BCKDHB, SLC16A1, HK2, LPCAT1 and PGR-AS1) were identified to construct the prognostic model of patients with EC. Based on GLRGs, the risk model's prognosis and independent prognostic value were established. The signature of GLRGs exhibited a robust correlation with the infiltration of immune cells and the sensitivity to drugs. In cytological experiments, we selected HK2 as candidate gene to verify its value in the occurrence and development of EC. Western blot and qRT-PCR revealed that HK2 was substantially expressed in EC cells. According to in vitro experiments, HK2 knockdown can increase EC cell apoptosis while suppressing EC cell migration, invasion, and proliferation. CONCLUSION: The GLRGs signature constructed in this study demonstrated significant prognostic value for patients with endometrial carcinoma, thereby providing valuable guidance for treatment decisions.

A novel TCGA-validated programmed cell-death-related signature of ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a gynecological malignancy tumor with high recurrence and mortality rates. Programmed cell death (PCD) is an essential regulator in cancer metabolism, whose functions are still unknown in OC. Therefore, it is vital to determine the prognostic value and therapy response of PCD-related genes in OC. METHODS: By mining The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Genecards databases, we constructed a prognostic PCD-related genes model and performed Kaplan-Meier (K-M) analysis and Receiver Operating Characteristic (ROC) curve for its predictive ability. A nomogram was created via Cox regression. We validated our model in train and test sets. Quantitative real-time PCR (qRT-PCR) was applied to identify the expression of our model genes. Finally, we analyzed functional analysis, immune infiltration, genomic mutation, tumor mutational burden (TMB) and drug sensitivity of patients in low- and high-risk group based on median scores. RESULTS: A ten-PCD-related gene signature including protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), 8-oxoguanine-DNA glycosylase (OGG1), HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 (HERC1), Caspase-2.(CASP2), Caspase activity and apoptosis inhibitor 1(CAAP1), RB transcriptional corepressor 1(RB1), Z-DNA binding protein 1 (ZBP1), CD3-epsilon (CD3E), Clathrin heavy chain like 1(CLTCL1), and CCAAT/enhancer-binding protein beta (CEBPB) was constructed. Risk score performed well with good area under curve (AUC) (AUC3 - year =0.728, AUC5 - year = 0.730). The nomogram based on risk score has good performance in predicting the prognosis of OC patients (AUC1 - year =0.781, AUC3 - year =0.759, AUC5 - year = 0.670). Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that the erythroblastic leukemia viral oncogene homolog (ERBB) signaling pathway and focal adhesion were enriched in the high-risk group. Meanwhile, patients with high-risk scores had worse OS. In addition, patients with low-risk scores had higher immune-infiltrating cells and enhanced expression of checkpoints, programmed cell death 1 ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO-1) and lymphocyte activation gene-3 (LAG3), and were more sensitive to A.443,654, GDC.0449, paclitaxel, gefitinib and cisplatin. Finally, qRT-PCR confirmed RB1, CAAP1, ZBP1, CEBPB and CLTCL1 over-expressed, while PPP1R15A, OGG1, CASP2, CD3E and HERC1 under-expressed in OC cell lines. CONCLUSION: Our model could precisely predict the prognosis, immune status and drug sensitivity of OC patients.

BCAR4 facilitates trastuzumab resistance and EMT in breast cancer via sponging miR-665 and interacting with YAP1.

Breast cancer antiestrogen resistance 4 (BCAR4) has been suggested that can modulate cell behavior, resulting in tumorigenesis and chemoresistance. However, the underlying mechanisms of BCAR4 in trastuzumab resistance (TR) is still elusive. Here, we explored the function and the underlying mechanism of BCAR4 involving in TR. We found that BCAR4 is significantly upregulated in trastuzumab-resistant BC cells. Knockdown of BCAR4 could sensitize the BC cells to trastuzumab and suppress epithelial-mesenchymal transition (EMT). Mechanically, BCAR4 promotes yes-associated protein 1 (YAP1) expression by competitively sponging miR-665, to activated TGF-ß signaling. Reciprocally, YAP1 could occupy the BCAR4 promoter to enhance its transcription, suggesting that there exists a positive feedback regulation between YAP1 and BCAR4. Targeting the BCAR4/miR-665/YAP1 axis may provide a novel insight of therapeutic approaches for TR in BC.

Dual Targeted Nanoparticles for the Codelivery of Doxorubicin and siRNA Cocktails to Overcome Ovarian Cancer Stem Cells.

Most anticancer treatments only induce the death of ordinary cancer cells, while cancer stem cells (CSCs) in the quiescent phase of cell division are difficult to kill, which eventually leads to cancer drug resistance, metastasis, and relapse. Therefore, CSCs are also important in targeted cancer therapy. Herein, we developed dual-targeted and glutathione (GSH)-responsive novel nanoparticles (SSBPEI-DOX@siRNAs/iRGD-PEG-HA) to efficiently and specifically deliver both doxorubicin and small interfering RNA cocktails (siRNAs) (survivin siRNA, Bcl-2 siRNA and ABCG2 siRNA) to ovarian CSCs. They are fabricated via electrostatic assembly of anionic siRNAs and cationic disulfide bond crosslinking-branched polyethyleneimine-doxorubicin (SSBPEI-DOX) as a core. Interestingly, the SSBPEI-DOX could be degraded into low-cytotoxic polyethyleneimine (PEI). Because of the enrichment of glutathione reductase in the tumor microenvironment, the disulfide bond (-SS-) in SSBPEI-DOX can be specifically reduced to promote the controlled release of siRNA and doxorubicin (DOX) in the CSCs. siRNA cocktails could specifically silence three key genes in CSCs, which, in combination with the traditional chemotherapy drug DOX, induces apoptosis or necrosis of CSCs. iRGD peptides and "sheddable" hyaluronic acid (HA) wrapped around the core could mediate CSC targeting by binding with neuropilin-1 (NRP1) and CD44 to enhance delivery. In summary, the multifunctional delivery system SSBPEI-DOX@siRNAs/iRGD-PEG-HA nanoparticles displays excellent biocompatibility, accurate CSC-targeting ability, and powerful anti-CSC ability, which demonstrates its potential value in future treatments to overcome ovarian cancer metastasis and relapse. To support this work, as exhaustive search was conducted for the literature on nanoparticle drug delivery research conducted in the last 17 years (2007-2023) using PubMed, Web of Science, and Google Scholar.

Identification of upregulated exosomal miRNAs between A2780 and A2780/DDP human ovarian cancer cells by high-throughput sequencing.

Exosomal miRNAs are known to play important roles in ovarian cancer and chemotherapeutic resistance. However, a systematic evaluation of characteristics of exosomal miRNAs involved in cisplatin resistance in ovarian cancer remains totally unclear. Exosomes (Exo-A2780, Exo-A2780/DDP) were extracted from cisplatin-sensitive cells (A2780) and cisplatin-resistant cells (A2780/DDP). Differential exosomal miRNA expression profiles were found by high-throughput sequencing (HTS). Target genes of the exo-miRNAs were predicted by using two online databases to increase the prediction accuracy. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were utilized to find biological relationships with chemoresistance. RT‒qPCR of three exosomal miRNAs was performed, and a protein‒protein interaction (PPI) network was established to identify the hub genes. The GDSC database was used to prove the association between hsa-miR-675-3p expression and the IC50 value. An integrated miRNA-mRNA network was constructed to predict miRNA-mRNA associations. The connection between hsa-miR-675-3p and ovarian cancer was discovered by immune microenvironment analyses. The upregulated exosomal miRNAs could regulate gene targets through signalling pathways such as the Ras, PI3K/Akt, Wnt, and ErbB pathways. GO and KEGG analyses indicated that the target genes were involved in protein binding, transcription regulator activity and DNA binding. The RT‒qPCR results were consistent with the HTS data, and the results of PPI network analysis suggested that FMR1 and CD86 were the hub genes. GDSC database analysis and construction of the integrated miRNA-mRNA network suggested that hsa-miR-675-3p was associated with drug resistance. Immune microenvironment analyses showed that hsa-miR-675-3p was crucial in ovarian cancer. The study suggested that exosomal hsa-miR-675-3p is a potential target for treating ovarian cancer and overcoming cisplatin resistance.

Dual-Activated Nano-Prodrug for Chemo-Photodynamic Combination Therapy of Breast Cancer.

Herein, we developed a dual-activated prodrug, BTC, that contains three functional components: a glutathione (GSH)-responsive BODIPY-based photosensitizer with a photoinduced electron transfer (PET) effect between BODIPY and the 2,4-dinitrobenzenesulfonate (DNBS) group, and an ROS-responsive thioketal linker connecting BODIPY and the chemotherapeutic agent camptothecin (CPT). Interestingly, CPT displayed low toxicity because the active site of CPT was modified by the BODIPY-based macrocycle. Additionally, BTC was encapsulated with the amphiphilic polymer DSPE-mPEG2000 to improve drug solubility and tumor selectivity. The resulting nano-prodrug passively targeted tumor cells through enhanced permeability and retention (EPR) effects, and then the photosensitizing ability of the BODIPY dye was restored by removing the DNBS group with the high concentration of GSH in tumor cells. Light-triggered ROS from activated BODIPY can not only induce apoptosis or necrosis of tumor cells but also sever the thioketal linker to release CPT, achieving the combination treatment of selective photodynamic therapy and chemotherapy. The antitumor activity of the prodrug has been demonstrated in mouse mammary carcinoma 4T1 and human breast cancer MCF-7 cell lines and 4T1 tumor-bearing mice.

The marine natural product, dicitrinone B, induces apoptosis through autophagy blockade in breast cancer.

Being a highly conserved catabolic process, autophagy is induced by various forms of cellular stress, and its modulation has considerable potential as a cancer therapeutic approach. In the present study, it was demonstrated that dicitrinone B (DB), a rare carbon­bridged citrinin dimer, may exert anticancer effects by blocking autophagy at a late stage, without disrupting lysosomal function in MCF7 breast cancer and MDA­MB­231 triple­negative breast cancer cells. Furthermore, it was discovered that DB significantly enhanced intracellular reactive oxygen species (ROS) production and that the removal of ROS was followed by the attenuation of autophagy inhibition. In addition, DB exerted notable inhibitory effects on the proliferation and promoting effects on the apoptosis of MCF7 and MDA­MB­231 cells. In combination with conventional chemotherapeutic drugs, DB exhibited a further enhanced synergistic effect than when used as a single agent. Overall, the data of the present study demonstrate that DB may prove to be a promising autophagy inhibitor with anticancer activity against breast cancer.

Advances in plant-derived natural products for antitumor immunotherapy.

In recent years, immunotherapy has emerged as a novel antitumor strategy in addition to traditional surgery, radiotherapy and chemotherapy. It uniquely focuses on immune cells and immunomodulators in the tumor microenvironment and helps eliminate tumors at the root by rebuilding the immune system. Despite remarkable breakthroughs, cancer immunotherapy still faces many challenges: lack of predictable and prognostic biomarkers, adverse side effects, acquired treatment resistance, high costs, etc. Therefore, more efficacious and efficient, safer and cheaper antitumor immunomodulatory drugs have become an urgent requirement. For decades, plant-derived natural products obtained from land and sea have provided the most important source for the development of antitumor drugs. Currently, more attention is being paid to the discovery of potential cancer immunotherapy modulators from plant-derived natural products, such as polysaccharides, phenols, terpenoids, quinones and alkaloids. Some of these agents have outstanding advantages of multitargeting and low side effects and low cost compared to conventional immunotherapeutic agents. We intend to summarize the progress of comprehensive research on these plant-derived natural products and their derivatives and discuss their possible mechanisms in regulating the immune system and their efficacy as monotherapies or in combination with regular chemotherapeutic agents.

A 13-Gene Metabolic Prognostic Signature Is Associated With Clinical and Immune Features in Stomach Adenocarcinoma.

Patients with advanced stomach adenocarcinoma (STAD) commonly show high mortality and poor prognosis. Increasing evidence has suggested that basic metabolic changes may promote the growth and aggressiveness of STAD; therefore, identification of metabolic prognostic signatures in STAD would be meaningful. An integrative analysis was performed with 407 samples from The Cancer Genome Atlas (TCGA) and 433 samples from Gene Expression Omnibus (GEO) to develop a metabolic prognostic signature associated with clinical and immune features in STAD using Cox regression analysis and least absolute shrinkage and selection operator (LASSO). The different proportions of immune cells and differentially expressed immune-related genes (DEIRGs) between high- and low-risk score groups based on the metabolic prognostic signature were evaluated to describe the association of cancer metabolism and immune response in STAD. A total of 883 metabolism-related genes in both TCGA and GEO databases were analyzed to obtain 184 differentially expressed metabolism-related genes (DEMRGs) between tumor and normal tissues. A 13-gene metabolic signature (GSTA2, POLD3, GLA, GGT5, DCK, CKMT2, ASAH1, OPLAH, ME1, ACYP1, NNMT, POLR1A, and RDH12) was constructed for prognostic prediction of STAD. Sixteen survival-related DEMRGs were significantly related to the overall survival of STAD and the immune landscape in the tumor microenvironment. Univariate and multiple Cox regression analyses and the nomogram proved that a metabolism-based prognostic risk score (MPRS) could be an independent risk factor. More importantly, the results were mutually verified using TCGA and GEO data. This study provided a metabolism-related gene signature for prognostic prediction of STAD and explored the association between metabolism and the immune microenvironment for future research, thereby furthering the understanding of the crosstalk between different molecular mechanisms in human STAD. Some prognosis-related metabolic pathways have been revealed, and the survival of STAD patients could be predicted by a risk model based on these pathways, which could serve as prognostic markers in clinical practice.

Survival-associated alternative splicing events interact with the immune microenvironment in stomach adenocarcinoma.

BACKGROUND: Alternative splicing (AS) increases the diversity of mRNA during transcription; it might play a role in alteration of the immune microenvironment, which could influence the development of immunotherapeutic strategies against cancer. AIM: To obtain the transcriptomic and clinical features and AS events in stomach adenocarcinoma (STAD) from the database. The overall survival data associated with AS events were used to construct a signature prognostic model for STAD. METHODS: Differentially expressed immune-related genes were identified between subtypes on the basis of the prognostic model. In STAD, 2042 overall-survival-related AS events were significantly enriched in various pathways and influenced several cellular functions. Furthermore, the network of splicing factors and overall-survival-associated AS events indicated potential regulatory mechanisms underlying the AS events in STAD. RESULTS: An eleven-AS-signature prognostic model (CD44|14986|ES, PPHLN1|21214|AT, RASSF4|11351|ES, KIAA1147|82046|AP, PPP2R5D|76200|ES, LOH12CR1|20507|ES, CDKN3|27569|AP, UBA52|48486|AD, CADPS|65499|AT, SRSF7| 53276|RI, and WEE1|14328|AP) was constructed and significantly related to STAD overall survival, immune cells, and cancer-related pathways. The differentially expressed immune-related genes between the high- and low-risk score groups were significantly enriched in cancer-related pathways. CONCLUSION: This study provided an AS-related prognostic model, potential mechanisms for AS, and alterations in the immune microenvironment (immune cells, genes, and pathways) for future research in STAD.

Reduction sensitive nanocarriers mPEG-g-γ-PGA/SSBPEI@siRNA for effective targeted delivery of survivin siRNA against NSCLC.

Poly γ-glutamic acid (γ-PGA) is attractive due to its desirable biological properties such as nontoxicity, excellent biocompatibility, and minimal immunogenicity. Additionally, γ-PGA could be recognized by γ-glutamyl transpeptidase, which is regarded as a potential biomarker for many tumors. In this study, we have developed a new biodegradable, reduction sensitive, and tumor-specific gene nano-delivery platform consisting of a cationic carrier (SSBPEI) for siRNA condensation, mPEG shell for nanoparticle stabilization, and γ-PGA for accelerated cellular uptake. Disulfide bonds (-SS-) could be reduced specifically in the tumor environment, which is full of reductants such as glutathione reductase. Conjugating polyethylene glycol (PEG) to the γ-PGA led to the formation of mPEG-g-γ-PGA, with a decreased positive charge on the surface of SSBPEI@siRNA and substantially higher stability in an aqueous medium. As a result, mPEG-g-γ-PGA/SSBPEI@siRNA nanoparticles could protect siRNAs from RNase A degradation and release siRNAs in a reduction sensitive way. The multifunctional delivery system was shown to silence the Survivin gene and further promote chemotherapeutic drug-induced apoptosis in the A549 NSCLC cell line efficiently, thereby representing a novel promising platform for the delivery of siRNAs.

4,4'­Bond secalonic acid D targets SP cells and inhibits metastasis in hepatocellular carcinoma.

The existence of cancer stem cells (CSCs) is considered to be the main reason for chemoresistance, metastasis and the ultimate failure of treatment in hepatocellular carcinoma (HCC). However, there are a few chemical agents that may inhibit CSCs. The present study identified that 4,4'­bond secalonic acid D (4,4'­SAD), a compound isolated from the marine­derived fungus Penicillium oxalicum, inhibited the growth of side population (SP) cells isolated from human liver cancer cell lines PLC/PRF/5 and HuH­7 by attenuating the expression of ATP­binding cassette superfamily G member 2. Furthermore, the results of wound healing, Transwell, western blotting and reverse transcription­quantitative PCR assays demonstrated that 4,4'­SAD suppressed the invasion and migration of SP cells by downregulating matrix metallopeptidase 9 (MMP­9) and upregulating the antagonist tissue inhibitor of metalloproteinases 1 in vitro. Moreover, in vivo study results found that 4,4'­SAD had anti­lung metastasis efficacy via the decrease of MMP­9 expression in the H22 HCC model of Kunming mice. Therefore, the present study identified the potential of 4,4'­SAD as a promising candidate for the treatment of advanced liver cancer.

Reduction sensitive CC9-PEG-SSBPEI/miR-148b nanoparticles: Synthesis, characterization, targeting delivery and application for anti-metastasis.

miRNAs such as miR-148b play crucial regulatory role in tumor metastasis, but their applications are limited because they are easy to degrade in serum conditions and lack targeting ability. Herein, CC9-PEG-SSBPEI was synthesized and used as nano-carrier for miR-148b. DLS and gel retardation analyses indicated that CC9-PEG-SSBPEI could combine with miR-148b by charge interaction and formed into nanoparticles with the size changed from 811.6 nm to 146.4 nm. CC9-PEG-SSBPEI could protect miR-148b from RNase A degradation and showed a reduction sensitive release of miR-148b. FACS analysis and CLSM images displayed that the conjugated CC9 peptide improved the accumulation and penetration of the nanoparticles in HuH-7 liver cancer cells through binding with the target of miR-148b neuropilin-1(NRP-1) on the cell surface. The raised level of miR-148b in turn inhibited the expression of NRP-1 and suppressed the migration of HuH-7 liver cancer cells. Moreover, hemolysis and cytotoxicity assay demonstrated that the nanoparticles had good hemo- and cyto- compatibility. Hence, CC9-PEG-SSBPEI/miR-148b nanoparticles had the potential for targeting delivery of miR-148b and anti-metastasis of hepatocellular carcinoma (HCC) cells.

Isolation of 4,4'-bond secalonic acid D from the marine-derived fungus Penicillium oxalicum with inhibitory property against hepatocellular carcinoma.

4,4'-bond secalonic acid D (4,4'-SAD) is a known compound isolated from the marine-derived fungus Penicillium oxalicum. No study about the antitumor effect of this compound has been reported, except for a few focusing on its bactericidal properties. Herein, we performed an in vitro biology test and found that 4,4'-SAD stimulated the apoptosis of tumor cells in the human hepatocellular carcinoma cell lines PLC/PRF/5 and HuH-7 by activating caspase-3, caspase-8, caspase-9, PARP, p53, and cyclin B1, as well as by regulating the Bax/Bcl-2 ratio. In vivo studies showed that 4,4'-SAD had antitumor efficacy in H22 cell xenograft model. Immunohistochemical analysis revealed that 4,4'-SAD could regulate Bax expression, which is a biomarker of tumor growth. In summary, 4,4'-SAD significantly inhibited tumor growth both in vivo and in vitro.

STAT3 antisense oligonucleotide AZD9150 in a subset of patients with heavily pretreated lymphoma: results of a phase 1b trial.

BACKGROUND: The Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin's lymphoma population, primarily consisting of patients with DLBCL. METHODS: Patients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells. RESULTS: Thirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance. CONCLUSIONS: AZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing. TRIAL REGISTRATION: Registered at ClinicalTrials.gov: NCT01563302 . First submitted 2/13/2012.

Comparative proteomics of side population cells derived from human hepatocellular carcinoma cell lines with varying metastatic potentials.

Metastasis and recurrence following surgery are major reasons for the high mortality rate and poor prognosis associated with hepatocellular carcinoma (HCC). Cancer stem cells (CSCs) are thought to be able to cause cancer, and to be the primary cause of tumor recurrence and metastasis. The underlying mechanisms of the metastatic potential of CSCs is poorly understood. In the present study, side population (SP) cells were isolated from 4 HCC cell lines, and their self-renewal and migratory abilities were compared. The results demonstrate that SP cells from different cell lines exhibited similar self-renewal abilities but different metastatic potentials. Furthermore, the overall proteomes of the SP cells were systematically quantified. This revealed 11 and 19 differentially expressed proteins (DEPs), upregulated and downregulated, respectively, associated with increased metastatic potential. These proteins were involved in the 'regulation of mRNA processing' and 'cytoskeleton organization' biological processes. The majority of the proteins were involved in 'cell proliferation', 'migration' and 'invasion of cancer', and may promote HCC metastasis in a synergistic manner. The AKT and nuclear factor-κB signaling pathways may contribute to the regulation of HCC metastasis through regulating the DEPs in SP cells. To the best of our knowledge, the present study is the first to demonstrate the overall proteome difference among SP cells from the different HCC cell lines with different metastatic potentials. The present study provides novel information regarding the metastatic potential of CSCs, which will facilitate further investigation of the topic.

Iso-pencillixanthone A from a marine-derived fungus reverses multidrug resistance in cervical cancer cells through down-regulating P-gp and re-activating apoptosis.

The occurrence of multidrug resistance (MDR) is highly associated with the overexpression of ATP-binding cassette (ABC) transporters, among which, P-glycoprotein (P-gp) plays one of the most important roles. Iso-pencillixanthone A (iso-PXA) is a compound isolated from the marine-derived fungus Penicillium oxalicum. No studies on the anti-tumor effect of this compound have been reported, except for a few focusing on its bactericidal properties. In this study, we found iso-PXA could stimulate P-gp ATPase activity and attenuate P-gp expression to increase the intracellular drug concentration in the cervical vincristine (VCR)-resistant cell line HeLa/VCR. Then, it increased ROS generation, depolarized MMP, promoted the release of cytochrome c from mitochondria, and further activated caspase-9, caspase-3 and PARP to induce cell apoptosis effectively through the intrinsic pathway. Caspase-8 medicated cleavage of Bid into the truncated form tBid partially initiated the mitochondrial apoptotic events. The elevation of the Bax/Bcl-2 ratio, the accumulation of FBW7 and the degradation of Mcl-1 accelerated the iso-PXA induced apoptotic process. The HeLa/VCR cell xenograft model again confirmed that iso-PXA had much better efficacy than vincristine in vivo. Taken together, these findings demonstrated that iso-PXA elicited remarkable anti-tumor and anti-MDR activity through inhibiting P-gp expression and function and re-activating the intrinsic apoptosis pathway in vitro and in vivo, suggesting it as a potential chemotherapeutic lead compound in the treatment of cervical MDR cancers.

[Construction of lentiviral vector of siRNA specific for γ-synuclein and its inhibition effect on colorectal carcinoma cell line SW1116].

OBJECTIVE: To construct a lentiviral vector carrying the γ-synuclein(SNCG) gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and then investigate the inhibition of the growth and invasion capacity of SW1116 cells. METHODS: RNA interference fragment was designed according to the SNCG sequence (GenBank: No.NM003087.2), and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP (RNAi group) and LV-SNCG-NC-EGFP (NC group) were constructed and packaged to produce lentivirus venom. The supernatants of different virus-producing cells were used to transfect SW1116 cells respectively. Wild SW1116 cells were used as blank control (CON group) EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively. RESULTS: Recombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirus could effectively infect SW1116 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV-SNCG-NC-EGFP was 8×10(8) TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression (1.009±0.161 vs. 0.114±0.030, P=0.009), and showed tremendous silencing effect as 76.8%(P<0.05). SNCG protein expression was also significantly reduced (RNAi:12.001±2.884, NC:32.443±4.731, CON:34.308±6.920, P<0.05). After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively(P=0.036). In soft agar assay, clones in RNAi group were smaller[RNAi:(0.582±0.103) mm, NC:(1.863±0.316) mm, CON:(1.749±0.525) mm]. Colony formation rate of RNAi group was down to (17.1±3.5)%, which was significantly lower than (36.5±4.3)% in NC group and (33.8±3.9)% in CON group. In migration test, the number of invasion cell was 37.4±9.3 in RNAi group, which was significantly less than 112.3±8.6 in NC group and 100±0.0 in CON group. CONCLUSION: Expression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.

Antitumor Effects and Related Mechanisms of Penicitrinine A, a Novel Alkaloid with a Unique Spiro Skeleton from the Marine Fungus Penicillium citrinum.

Penicitrinine A, a novel alkaloid with a unique spiro skeleton, was isolated from a marine-derived fungus Penicillium citrinum. In this study, the isolation, structure and biosynthetic pathway elucidation of the new compound were described. This new compound showed anti-proliferative activity on multiple tumor types. Among them, the human malignant melanoma cell A-375 was confirmed to be the most sensitive. Morphologic evaluation, apoptosis rate analysis, Western blot and real-time quantitative PCR (RT-qPCR) results showed penicitrinine A could significantly induce A-375 cell apoptosis by decreasing the expression of Bcl-2 and increasing the expression of Bax. Moreover, we investigated the anti-metastatic effects of penicitrinine A in A-375 cells by wound healing assay, trans-well assay, Western blot and RT-qPCR. The results showed penicitrinine A significantly suppressed metastatic activity of A-375 cells by regulating the expression of MMP-9 and its specific inhibitor TIMP-1. These findings suggested that penicitrinine A might serve as a potential antitumor agent, which could inhibit the proliferation and metastasis of tumor cells.

miRNA-148b suppresses hepatic cancer stem cell by targeting neuropilin-1.

The existence of cancer stem cells (CSCs) is considered as a direct reason for the failure of clinic treatment in hepatocellular carcinoma (HCC). Growing evidences have demonstrated that miRNAs play an important role in regulation of stem cell proliferation, differentiation and self-renewal and their aberrances cause the formation of CSCs and eventually result in carcinogenesis. We recently identified miRNA-148b as one of the miRNAs specifically down-regulated in side population (SP) cells of PLC/PRF/5 cell line. However, it remains elusive how miRNA-148b regulates CSC properties in HCC. In the present study, we observed that overexpression or knockdown of miR-148b through lentiviral transfection could affect the proportion of SP cells as well as CSC-related gene expression in HCC cell lines. In addition, miR-148b blocking could stimulate cell proliferation, enhance chemosensitivity, as well as increase cell metastasis and angiogenesis in vitro. More importantly, miR-148b could significantly suppress tumorigenicity in vivo. Further studies revealed that Neuropilin-1 (NRP1), a transmembrane co-receptor involved in tumour initiation, metastasis and angiogenesis, might be the direct target of miRNA-148b. Taking together, our findings define that miR-148b might play a critical role in maintenance of SP cells with CSC properties by targeting NRP1 in HCC. It is the potential to develop a new strategy specifically targeting hepatic CSCs (HCSCs) through restoration of miR-148b expression in future therapy.