Elesclomol Loaded Copper Oxide Nanoplatform Triggers Cuproptosis to Enhance Antitumor Immunotherapy.

The induction of cuproptosis, a recently identified form of copper-dependent immunogenic cell death, is a promising approach for antitumor therapy. However, sufficient accumulation of intracellular copper ions (Cu2+) in tumor cells is essential for inducing cuproptosis. Herein, an intelligent cuproptosis-inducing nanosystem is constructed by encapsulating copper oxide (CuO) nanoparticles with the copper ionophore elesclomol (ES). After uptake by tumor cells, ES@CuO is degraded to release Cu2+ and ES to synergistically trigger cuproptosis, thereby significantly inhibiting the tumor growth of murine B16 melanoma cells. Moreover, ES@CuO further promoted cuproptosis-mediated immune responses and reprogrammed the immunosuppressive tumor microenvironment by increasing the number of tumor-infiltrating lymphocytes and secreted inflammatory cytokines. Additionally, combining ES@CuO with programmed cell death-1 (PD-1) immunotherapy substantially increased the antitumor efficacy in murine melanoma. Overall, the findings of this study can lead to the use of a novel strategy for cuproptosis-mediated antitumor therapy, which may enhance the efficacy of immune checkpoint inhibitor therapy.

Interferon-Gamma-Inducible Protein 16 Inhibits Hepatocellular Carcinoma via Interferon Regulatory Factor 3 on Chemosensitivity.

BACKGROUND AND AIM: Previous reports have suggested IFI16 as a tumor suppressor in hepatocellular carcinoma (HC). Nonetheless, the biological significance of IFI16 and its mechanism concerning resistance to cisplatin (DDP) in HC requires further exploration. METHODS: Samples of tumor and corresponding para-carcinoma tissues were acquired from patients with HC. Furthermore, DDP-resistant cell lines of HC, specifically HCC, Huh7 and Hepatoblastoma, HepG3, were generated by gradually increasing the concentration of DDP. Cell apoptosis and DNA damage were evaluated by utilizing flow cytometry assay and TUNEL staining. The interaction between IFI16 and interferon regulatory factor 3 (IRF3) proteins were analyzed using Co-Immunoprecipitation (Co-IP) assay. In vivo assays were conducted by establishing HC subcutaneous xenograft tumor models. RESULTS: The study found a reduction in IFI16 expression in both HC tissues and DDP-resistant HC cell lines. The binding of IFI16 to IRF3 regulated DNA damage-associated markers in vitro. Overexpression of IFI16 heightened the susceptibility of DDP-induced apoptosis and DNA damage, which was counteracted by IRF3 knockdown, while strengthened by IRF3 overexpression. Moreover, overexpression of IFI16 diminished in vivo DDP-resistant HC tumorigenicity. CONCLUSION: In summary, our findings suggest that IFI16 serves as a tumor suppressor in HC by promoting DNA damage via its interaction with IRF3, thereby reversing DDP resistance.

ASAP1 activates the IQGAP1/CDC42 pathway to promote tumor progression and chemotherapy resistance in gastric cancer.

Abnormal expression and remodeling of cytoskeletal regulatory proteins are important mechanisms for tumor development and chemotherapy resistance. This study systematically analyzed the relationship between differential expression of cytoskeleton genes and prognosis in gastric cancer (GC). We found the Arf GTP-activating protein ASAP1 plays a key role in cytoskeletal remodeling and prognosis in GC patients. Here we analyzed the expression level of ASAP1 in tissue microarrays carrying 564 GC tissues by immunohistochemistry. The results showed that ASAP1 expression was upregulated in GC cells and can be served as a predictor of poor prognosis. Moreover, ASAP1 promoted the proliferation, migration, and invasion of GC cells both in vitro and in vivo. We also demonstrated that ASAP1 inhibited the ubiquitin-mediated degradation of IQGAP1 and thus enhanced the activity of CDC42. The activated CDC42 upregulated the EGFR-MAPK pathway, thereby promoting the resistance to chemotherapy in GC. Taken together, our results revealed a novel mechanism by which ASAP1 acts in the progression and chemotherapy resistance in GC. This may provide an additional treatment option for patients with GC.

Tumor-infiltrating B cell is associated with the control of progression of gastric cancer.

This study aimed to further explore the clinicopathological correlation of B cell infiltration in gastric cancer (GC) and its impact on prognostic. By immunohistochemical method, CD20+ B cells, CD3+ T cells, CD66b+ tumor-associated neutrophils, CD163+ tumor-associated macrophages, and CD57+ natural killer cells were analyzed in consecutive sections of 584 GC tissues and 69 normal adjacent tissues. Kaplan-Meier and Cox regression analyses determined the relationship between clinical relevance or prognosis and B cell infiltration. The correlation between total B cell infiltration and various T cell subtype infiltration in GC tissues from 407 patients in the TCGA data was also analyzed. Kaplan-Meier and Cox regression analyses determined the effects of total B cell infiltration and various B cell subtype infiltration on the prognosis of patients with GC. The infiltration level of CD20+ B cells was positively correlated with that of T cells (risk ratio [RR] = 0.0930), especially CD4+ T cells and CD8+ T cells (P < 0.05). A high level of CD20+ B cell infiltration was significantly associated with low lymph node involvement and low TNM stage (P < 0.05). High levels of CD20+ B cell infiltration were significantly associated with improvements in overall survival and disease-free survival. Univariate Cox regression and multivariate Cox regression analysis showed that CD20+ B cell infiltration was an independent protective factor of prognosis. Higher levels of class-switched memory B cell and plasma cell also reflected better overall survival, and class-switched memory B cell and plasma cell were independent protective factors for prognosis. The findings indicate that B cell infiltration in GC, especially switched memory B cells and plasma cells, has a significant effect on tumor progression and prognosis.

Tumor-Infiltrating Immune Cells Act as a Marker for Prognosis in Colorectal Cancer.

Tumor-infiltrating immune cells (TIICs) play essential roles in cancer development and progression. However, the association of TIICs with prognosis in colorectal cancer (CRC) patients remains elusive. Infiltration of TIICs was assessed using ssGSEA and CIBERSORT tools. The association of TIICs with prognosis was analyzed in 1,802 CRC data downloaded from the GEO (https://www.ncbi.nlm.nih.gov/geo/) and TCGA (https://portal.gdc.cancer.gov/) databases. Three populations of TIICs, including CD66b+ tumor-associated neutrophils (TANs), FoxP3+ Tregs, and CD163+ tumor-associated macrophages (TAMs) were selected for immunohistochemistry (IHC) validation analysis in 1,008 CRC biopsies, and their influence on clinical features and prognosis of CRC patients was analyzed. Prognostic models were constructed based on the training cohort (359 patients). The models were further tested and verified in testing (249 patients) and validation cohorts (400 patients). Based on ssGSEA and CIBERSORT analysis, the correlation between TIICs and CRC prognosis was inconsistent in different datasets. Moreover, the results with disease-free survival (DFS) and overall survival (OS) data in the same dataset also differed. The high abundance of TIICs found by ssGSEA or CIBERSORT tools can be used for prognostic evaluation effectively. IHC results showed that TANs, Tregs, TAMs were significantly correlated with prognosis in CRC patients and were independent prognostic factors (PDFS ≤ 0.001; POS ≤ 0.023). The prognostic predictive models were constructed based on the numbers of TANs, Tregs, TAMs (C-indexDFS&OS = 0.86; AICDFS = 448.43; AICOS = 184.30) and they were more reliable than traditional indicators for evaluating prognosis in CRC patients. Besides, TIICs may affect the response to chemotherapy. In conclusion, TIICs were correlated with clinical features and prognosis in patients with CRC and thus can be used as markers.

Long Non-Coding RNA Differentiation Antagonizing Nonprotein Coding RNA (DANCR) Promotes Proliferation and Invasion of Pancreatic Cancer by Sponging miR-214-5p to Regulate E2F2 Expression.

BACKGROUND Long non-coding RNA differentiation antagonizing nonprotein coding RNA (lncRNA DANCR) has been reported to act as an oncogene in various human cancers. The role of DANCR in development of pancreatic cancer (PC) is unknown. The aim of our research was to investigate the biological role of DANCR in PC. MATERIAL AND METHODS Expressions of DANCR, miR-214-5p, and E2F2 mRNA in PC tissues and cell lines were examined by qRT-PCR. Western blotting was carried out for detection of E2F2 protein expression in PC cells. Transwell assays were used to examine the metastatic ability of PC cells, while CCK-8 and colony formation assay were applied to evaluate cell proliferation. The effects of DANCR on PC cells were assessed by knockdown in vitro and in vivo. The regulatory mechanism of competitive endogenous RNAs were obtained from bioinformatics prediction and luciferase reporter assay. RESULTS DANCR was markedly upregulated in clinical tissues and cell lines of PC. High DANCR expression exhibited a significant correlation with poor prognosis. DANCR knockdown inhibited growth and metastasis of PC cells. Furthermore, DANCR acted as sponge to regulate miR-214-5p, and miR-214-5p inhibitor reversed the effects of DANCR knockdown on PC cells. Moreover, DANCR positively modulated E2F2 expression through miR-214-5p in PC cells. CONCLUSIONS Collectively, our findings demonstrated that lncRNA DANCR/miR-214-5p/E2F2 axis acts as an oncogene in PC development, which might provide a potential target for PC therapy.

IFI16 restoration in hepatocellular carcinoma induces tumour inhibition via activation of p53 signals and inflammasome.

OBJECTIVE: Interferon-inducible 16 (IFI16) is known to involve in p53-dependent tumour suppression and also the formation of inflammasome, which function, however, remains controversy during carcinogenesis as a pattern recognition receptor for tumour death-derived free DNA. In this study, we investigated the anti-tumour role of IFI16 in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Hepatocellular carcinoma tissues (n = 20) and corresponding non-neoplastic tissues (n = 20) were collected to determine the expression of IFI16. After the transfection of pcDNA3.1-IFI16 into Huh7 and SMMC7721 cells in vitro, the influence of IFI16 overexpression on cell vitality, colony formation, apoptosis and migration were analysed. The role effect of IFI16 in vivo was further investigated. RESULTS: The expression of IFI16 was significantly decreased in tumour tissues and cell lines. Overexpression of IFI16 induced decrease of cell vitality, colony formation and increased apoptosis with impaired ability of migration. Mechanistically, IFI16 could activate p53 at Ser15 to up-regulate the p21WAF1/CIP1 level to inhibit tumour growth and migration, which was restored by the p53 inhibitor Pifithrin-α (20 µmol/L). Moreover, IFI16-induced tumour cell death promoted the recruitment of inflammasome complex to enhance tumour inhibition, but the caspase-1 inhibitor Ac-YVAD-CMK (50 µmol/L) could suppress this process in HCC. The results in vivo indicated that restored expression of IFI16 in tumour cells effectively promote tumour regression, which could be partly abrogated by the inhibition of activation of p53 signals or induced inflammasome. CONCLUSION: IFI16 is a tumour suppressor in HCC via activation of p53 signals and inflammasome.

Transcriptomic analysis of gene expression profiles of stomach carcinoma reveal abnormal expression of mitotic components.

AIMS: In order to explore the etiology of gastric cancer on global gene expression level, we developed advanced bioinformatic analysis to investigate the variations of global gene expression and the interactions among them. MAIN METHODS: We downloaded the dataset GSE63288 from Gene Expression Omnibus (GEO) database which included 22 human gastric cancer and 22 healthy control samples. We identified the differential expression genes, and explored the Gene ontology (GO) and pathways of the differentially expressed genes. Furthermore, integrative interaction network and co-expression network were employed to identify the key genes which may contribute to gastric cancer progression. KEY FINDINGS: The results indicated that 5 kinases including BUB1, TTK protein kinase, Citron Rho-interacting kinase (CIT), ZAK and NEK2 were upregulated in gastric cancer. Interestingly, BUB1, TTK, CIT and NEK2 have shown high expression similarities and bound with each other, and participated in multiple phases of mitosis. Moreover, a subnet of co-expression genes e.g. KIF14, PRC1, CENPF and CENPI was also involved in mitosis which was functionally coupled with the kinases above. By validation assays, the results indicated that CIT, PRC1, TTK and KIF14 were significantly upregulated in gastric cancer. SIGNIFICANCE: These evidences have suggested that aberrant expression of these genes may drive gastric cancer including progression, invasion and metastasis. Although the causal relationships between gastric cancer and the genes are still lacking, it was reasonable to take them as biomarkers for diagnosis of gastric cancer.

Antitumor activity of emodin against pancreatic cancer depends on its dual role: promotion of apoptosis and suppression of angiogenesis.

BACKGROUND: Emodin has been showed to induce apoptosis of pancreatic cancer cells and inhibit tumor growth in our previous studies. This study was designed to investigate whether emodin could inhibit the angiogenesis of pancreatic cancer tissues and its mechanism. METHODOLOGY/PRINCIPAL FINDING: In accordance with our previous study, emodin inhibited pancreatic cancer cell growth, induced apoptosis, and enhanced the anti-tumor effect of gemcitabine on pancreatic caner cells in vitro and in vivo by inhibiting the activity of NF-κB. Here, for the first time, we demonstrated that emodin inhibited tumor angiogenesis in vitro and in implanted pancreatic cancer tissues, decreased the expression of angiogenesis-associated factors (NF-κB and its regulated factors VEGF, MMP-2, MMP-9, and eNOS), and reduced eNOS phosphorylation, as evidenced by both immunohistochemistry and western blot analysis of implanted tumors. In addition, we found that emodin had no effect on VEGFR expression in vivo. CONCLUSIONS/SIGNIFICANCE: Our results suggested that emodin has potential anti-tumor effect on pancreatic cancer via its dual role in the promotion of apoptosis and suppression of angiogenesis, probably through regulating the expression of NF-κB and NF-κB-regulated angiogenesis-associated factors.

Enhanced antitumor efficacy of gemcitabine by evodiamine on pancreatic cancer via regulating PI3K/Akt pathway.

Evodiamine has therapeutic potential against cancers. This study was designed to investigate whether combination therapy with gemcitabine and evodiamine enhanced antitumor efficacy in pancreatic cancer. In vitro application of the combination therapy triggered significantly higher frequency of pancreatic cancer cells apoptosis, inhibited the activities of PI3K, Akt, PKA, mTOR and PTEN, and decreased the activation of NF-κB and expression of NF-κB-regulated products. In vivo application of the combination therapy induced significant enhancement of tumor cell apoptosis, reductions in tumor volume, and inhibited activation of mTOR and PTEN. In conclusion, evodiamine can augment the therapeutic effect of gemcitabine in pancreatic cancer through direct or indirect negative regulation of the PI3K/Akt pathway.

Emodin reverses gemcitabine resistance in pancreatic cancer cells via the mitochondrial apoptosis pathway in vitro.

Gemcitabine resistance is a common problem of pancreatic cancer chemotherapy, and how to reverse it plays an important role in the treatment of pancreatic cancer. This study investigated the effect of emodin on the gemcitabine-resistant pancreatic cancer cell line SW1990/Gem, and explored the potential mechanism of its action. SW1990/Gem was obtained by culture of the pancreatic cancer cell line SW1990 in vitro by intermittently increasing the concentration of gemcitabine in the culture medium for 10 months, observing the morphology using inverted microscopy. SW1990/Gem cells were pretreated with emodin (10 µM) for different periods followed by treatment with gemcitabine (20 µM) for 48 h; cell proliferation was tested by MTT assay. SW1990/Gem cells were treated by emodin with different concentrations for 48 h, cell apoptosis was detected by flow cytometry (FCM). The expression of gene and protein, such as MDR-1 (P-gp), NF-κB, Bcl-2, Bax, cytochrome-C (cytosol), caspase-9 and -3 were measured by RT-PCR and Western blotting. The function of P-gp in SW1990/Gem cells was checked by FCM. The results showed that the SW1990/Gem cells changed greatly in morphology and the resistance index was 48.63. Emodin promoted cell apoptosis of the gemcitabine-resistant cell line SW1990/Gem in a dose-dependent manner. Emodin enhanced the SW1990/Gem cell sensitivity to gemcitabine in a time-dependent manner. Emodin monotherapy or combination with gemcitabine both decreased the gene and protein expression levels of MDR-1 (P-gp), NF-κB and Bcl-2 and inhibited the function of P-gp, but increased the expression levels of Bax, cytochrome-C (cytosol), caspase-9 and -3, and promoted cell apoptosis. This demonstrated that emodin had a reversing effect on the gemcitabine-resistant cell line SW1990/Gem, possibly via decreasing the function of P-gp and activating the mitochondrial apoptosis pathway in vitro.

Antitumor and apoptosis-promoting properties of emodin, an anthraquinone derivative from Rheum officinale Baill, against pancreatic cancer in mice via inhibition of Akt activation.

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.

Enhanced antitumor efficacy by the combination of emodin and gemcitabine against human pancreatic cancer cells via downregulation of the expression of XIAP in vitro and in vivo.

XIAP and NF-κB play an important role in chemotherapy resistance in pancreatic cancer. The purpose of this study was to explore the role of XIAP and NF-κB in potentiating the antitumor effect of gemcitabine by emodin in pancreatic cancer. SW1990 cells were treated by sodium chloride, gemcitabine, emodin or their combination (gemcitabine plus emodin). Cellular proliferation and apoptosis were detected by Cell Counting kit-8 (CCK-8) assay and flow cytometry in vitro. The combination therapy more significantly inhibited SW1990 cell growth and induced a higher percentage of apoptosis than monotherapy. Gemcitabine upregulated the expression of XIAP and NF-κB, while emodin or emodin plus gemcitabine downregulated them compared to the control group in vitro. SW1990 cells were used to establish orthotopic pancreatic tumor models in nude mice. Tumor-bearing mice were treated with sodium chloride, emodin, gemcitabine or their combination. After being treated for 4 weeks, the nude mice were imaged with high-resolution positron emission tomography (microPET) and fluorine-18-labeled fluorodeoxyglucose (18F-FDG) to detect the tumor/non-tumor ratio (T/NT ratio) and standard uptake value (SUV). The mice were sacrificed to determine tumor weight. The combination of emodin and gemcitabine showed more significant reduction in the T/NT ratio, SUV and tumor weight compared to monotherapy. The mRNA levels and the protein expression of XIAP and NF-κB were upregulated in the gemcitabine group, while they were downregulated in the emodin group and the combination group in vivo. Ki-67 prolif-eration index and TUNEL assay results also showed that emodin enhanced tumor apoptosis induced by gemcitabine in vivo. This study suggests that emodin enhances the antitumor effect of gemcitabine in SW1990 pancreatic cancer in vitro and in vivo, which may be via the downregulation of NF-κB expression, thus inhibiting the expression of XIAP.

Emodin prolongs recipient survival time after orthotopic liver transplantation in rats by polarizing the Th1/Th2 paradigm to Th2.

Advances in immunosuppressive drugs have improved the short-term survival of liver transplantation. However, drug toxicities have been a serious problem in patients after long-term administration. Therefore, it is necessary to develop a novel immunosuppressant with low-toxicity. We investigated the immunosuppressive effects of Emodin on acute graft rejection following liver transplantation in rats. The recipient rats of orthotopic liver transplantation were divided into groups as follows: isograft+NS group, allograft+NS group, and allograft+emodin group. The survival time of the recipients in each group was recorded. Histopathological changes in the liver, as well as serum concentrations of IL-2, TNF-α, and IL-10 and their expressions in liver tissue were determined. Our results showed that Emodin treatment prolonged liver allograft survival time and inhibited histopathologic changes of acute graft rejection. The rejection activity index in groups isograft+NS, allograft+NS, and allograft+emodin were 1.52 ± 0.37, 6.95 ± 0.75, and 4.23 ± 0.51, respectively (P < 0.01, isograft+NS group vs. allograft+emodin group and allograft+NS group vs. allograft+emodin group). The serum levels of IL-2 and TNF-α were down-regulated but that of IL-10 was up-regulated by Emodin. Serum levels of IL-2 and TNF-α were higher in allograft+NS group than the allograft+emodin group, but that of IL-10 showed opposite effects (P < 0.05 or 0.01). Changes in the expression of these cytokines in transplanted liver tissue were consistent with changes in serum concentrations. These results demonstrate that Emodin has therapeutic potentials for alleviating acute rejection following liver transplantation in rats and prolonging liver allograft survival. The mechanisms underlying this effect may be associated with polarizing the Th1/Th2 paradigm to Th2.

Prevention and treatment of lymphorrhoea following surgery for gastric cancer.

BACKGROUND: Lymphorrhoea is a rare complication of abdominal surgery. However, there have been a few reports of lymphorrhoea following radical gastrectomy. Here, we retrospectively review the clinical analysis and treatment of lymphorrhoea based on our experiences. METHODS: We retrospectively reviewed a total of 1596 patients who underwent surgery for gastric cancer between January 1995 and January 2007. D1 and D2 lymphadenectomies were performed in 1104 patients, and D3 and D4 lymphadenectomies were performed in the other 492 patients. Disrupted lymph vessels were ligated in 545 patients, and electrically cauterized in 559 patients. Before December 31 2000, total parenteral nutrition (TPN) was administered to all the patients, and after 1 January 2001, TPN was supplemented with octreotide in all the post-operative patients. RESULTS: The incidence of lymphorrhoea in patients with D3 and D4 lymphadenectomy was much higher than that in D1 and D2 lymphadenectomy patients (P < 0.05). In addition, the incidence of lymphorrhoea in patients in whom the electrotome cautery was significantly higher than that in patients who received ligation. The addition of octreotide to TPN can reduce the quantity and duration of lymphorrhoea (P < 0.05). CONCLUSION: Ligating rather than cauterizing the disrupted lymph vessels can be done to minimize the incidence of lymphorrhoea. The combination of Octreotide and TPN appears to be an effective therapeutic modality for lymphorrhoea.