H22 Cells Research Articles

Extraction, characterization, and in vivo antitumor activity of a novel polysaccharide from Coriandrum sativum L.

A novel polysaccharide was extracted from Coriandrum sativum L. at a yield of 4.56 ± 0.17% (n= 3). The extraction was optimized using response surface methodology: powder-to-liquid ratio 1:21 g/ml, extraction time 188 min, temperature 81°C, and three replicate extractions. The purified polysaccharide had an average molecular weight of 1.30 × 106 Da and was composed of rhamnose, arabinose, galactose, glucose, and galacturonic acid in molar ratios of 1.52: 8.14: 20.85: 1: 2.42 with α-L-Araf-(1→, →6)-β-D-Galp-(1→, →4)-α-GalpA-(1→ and →2, 4)-α-Rhap-(1→). In vivo tests demonstrated that the polysaccharide suppressed H22 tumor growth in mice and protected the immune organs. Annexin V-FITC/PI, PI, and JC-1 staining showed that the primary mechanism of tumor inhibition was the induction of apoptosis and S-phase arrest with apoptosis achieved via a mitochondrial pathway. PRACTICAL APPLICATIONS: Coriandrum sativum L. is used as a culinary spice but its medicinal value has also been widely recognized. A novel polysaccharide was extracted from this herbaceous plant and its structure and bioactivity were investigated. This high-molecular-weight polysaccharide exhibited antitumor effects against H22 cells in mice and had potential to be developed as an anti-liver cancer medicine and functional food supplement.

An Acute Inflammation with Special Expression of CD11 & CD4 Produces Abscopal Effect by Intramoral Injection Chemotherapy Drug with Hapten in Animal Model

Aim: To study the immunity reaction in tumor by intratumoral injection with a drug PYM and DNP, where it produces abscopal effect by the expression of immunological genes of tumor on home side (left side) while it brings a similar expression of same genes in tumor on opposite side (right side) of mice. Method: Prepare each tumor on left side of 6 mice and injected intratumoral with a PYM+DNP and PYM control on day 1 and 7. Two day later of day 1, one of groups mice were inoculated with 0.2ml of H22 cells (105/ml) again on the right underarms as opposite side for controls. On day15, the tumor on bilateral sides were excised for qPCR measurement. Result：It showed that inflammation with the expression of the CoL1a1, CD4, IL12aÂ, TGFb1Â, Elastin, Elastin, Cox2, CD11b/c, CD8, TNFa in different groups; The inflammation in both side of tumor but these is not only a increasing expression of Collal, CD4, IL12aÂ, TGFb1Â, Elastin, NFKB, Cox2, CD11c, CD8 and TNFa in tumor on home side of mice treated with PYM+DNP but also a similar an increasing expression of same genes in tumor on opposite side of mice which not treated at all. In the control group, it showed that inflammation without an expression of all factors related above immunity genes in both tumor on home treated with PYM only and opposite side of mice which not treated at all. Conclusion: It indicated that PYM and hapten of DNP can induce an inflammation with stimulation of immunity reaction with the expression of the CoL1a1, CD4, IL12aÂ, TGFb1Â, Elastin, Elastin, Cox2, CD11b/c, CD8, TNFaÂ, which resulted in an abscopale effect. PYM can induce an inflammation but without expression of immuno genes, therefore, hapten is playing an important role with PYM in the special immunity reaction.

Therapeutic effect of Panax notoginseng saponins combined with cyclophosphamide in mice bearing hepatocellular carcinoma H22 cell xenograft

To investigate the therapeutic effects of total saponins from Panax notognseng (PNS) combined with cyclophosphamide (CTX) in mice bearing hepatocellular carcinoma H22 cell xenograft. We examined the effects of treatment with different concentrations of PNS on H22 cell proliferation for 24 to 72 h in vitro using CCK8 colorimetric assay. Annexin V/PI double fluorescence staining was used to detect the effect of PNS on apoptosis of H22 cells. Mouse models bearing H22 cell xenograft were established and treated with CTX (25 mg/kg), PNS (120, 240 or 480 mg/kg), alone or in combinations. After treatments for consecutive 10 days, the mice were euthanized for examinations of carbon clearance ability of the monocytes and macrophages, splenic lymphocyte proliferation, tumor necrosis factor (TNF-α), interleukin-2 (IL-2), serum hemolysin antibody level, blood indicators, and the tumor inhibition rate. Treatment with PNS concentration-dependently inhibited the proliferation and significantly promoted apoptosis of cultured H22 cells (P < 0.01). In the tumor-bearing mouse models, PNS alone and its combination with CTX both resulted in obvious enhancement of phagocytosis of the monocyte-macrophages, stimulated the proliferation of splenic lymphocytes, promoted the release of TNF-α and IL-2 and the production of serum hemolysin antibody, and increased the number of white blood cells, red blood cells and lymphocytes in the peripheral blood. Treatment with 480 mg/kg PNS combined with CTX resulted in a tumor inhibition rate of 83.28% (P < 0.01) and a life prolonging rate of 131.25% in the mouse models (P < 0.05). PNS alone or in combination with CTX can improve the immunity and tumor inhibition rate and prolong the survival time of H22 tumor-bearing mice.

Structural characterization of a low molecular weight Bletilla striata polysaccharide and antitumor activity on H22 tumor-bearing mice.

In this study, a novel low molecular weight polysaccharide (named LMW-BSP) was extracted from Bletilla striata at 4°C. The results of structural characteristics analysis showed that LMW-BSP was a 23kDa neutral polysaccharide contained glucose and mannose at a molar ratio of 1.00:1.26. Structural investigations of the periodate oxidation studies, Smith-degradation as well as methylation were performed, and combined with 1D and 2D NMR spectroscopy, the main chain residues sequence of LMW-BSP was concluded to be: α-D-Manp-(1→3)-β-D-Manp-(1→[4)-β-D-Glcp-(1]2→4)-β-D-Manp-(1→3)-β-D-Manp-(1→. Moreover, the antitumor activity of LMW-BSP was evaluated in H22 tumor-bearing mice. And the results suggested that LMW-BSP could effectively improve immune cells activities and lymphocytes subsets proportions dose-dependently in tumor-bearing mice, leading to the apoptosis of H22 cells via G1 phase arrested. LMW-BSP inhibited tumor growth and exhibited antitumor effects in vivo. And it supported considering the novel polysaccharide as a potential drug component in hepatocellular carcinoma treatment.

Long non-coding RNA Opa interacting protein 5-antisense RNA 1 binds to micorRNA-34a to upregulate oncogenic PD-L1 in non-small cell lung cancer

ABSTRACT Long non-coding RNA (lncRNA) OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) plays an oncogenic role in several types of cancer, but whether it is involved in non-small-cell lung cancer (NSCLC) is unclear. Our preliminary sequencing analysis revealed the upregulation of OIP5-AS1 in NSCLC. In this study, gene expression levels were analyzed by RT-qPCR. RNA-RNA pull-down assay was applied to detect direct interactions between RNAs. Overexpression assays were performed to explore the relationship between miR-34a and OIP5-AS1. CCK-8 assay and colony formation assay were applied to evaluate cell proliferation. In NSCLC cells (H23), overexpression of OIP5-AS1 increased the expression levels of programmed death-ligand 1 (PD-L1). In addition, inhibition of OIP5-AS1 and overexpression of miR-34a decreased the expression levels of PD-L1, and miR-34a significantly blocked the role of overexpression of OIP5-AS1. Overexpression of OIP5-AS1 and PD-L1 promoted H23 and H22 cells proliferation, while silencing of miR-34a and OIP5-AS1 played opposite roles and eliminated the effects of overexpression of OIP5-AS1 on cell proliferation. Therefore, OIP5-AS1 was upregulated to enhance the expression of oncogenic PD-L1 by sponging miR-34a in NSCLC, leading to promoted NSCLC cell proliferation. Our study also demonstrated that OIP5-AS1 was upregulated while miR-34a was downregulated in NSCLC.

Suppression of the transforming growth factor-β signaling pathway produces a synergistic effect of combination therapy with programmed death receptor 1 blockade and radiofrequency ablation against hepatic carcinoma in mice

ABSTRACT Primary liver cancer (PLC) significantly affects the health of patients globally owing to its high morbidity and low survival rate. Radiofrequency ablation (RFA) has recently been introduced for the clinical treatment of PLC. However, significant immunosuppressive effects are induced by RFA, which limits its application. This study aimed to explore the potential of combination therapy with RFA by investigating the effects of siRNAs against programmed death receptor 1 (PD-1) and transforming growth factor-β (TGF-β) on the antitumor effect induced by RFA. We observed that compared with si-NC, cell viability was reduced, apoptosis rate was elevated, release of inflammatory factors and percentage of CD3+CD8+ cells were increased, and the PI3K/AKT/mTOR pathway was repressed in the co-culture of RFA-treated H22 cells and CD8+ T cells by transfection with si-PD-1 and si-TGF-β; these effects were further enhanced by co-transfection with si-PD-1 and si-TGF-β. Additionally, in H22 cell xenograft-bearing mice treated with RFA, compared with the si-NC group, repressed tumor growth, prolonged survival, increased production of inflammatory factors and expression of CD3 and CD8 in tumor tissues, and downregulation of the PI3K/AKT/mTOR pathway were observed in the si-PD-1 and si-TGF-β groups; these effects were further enhanced in the si-PD-1 + si-TGF-β group. Taken together, our data revealed that suppression of the TGF-β signaling pathway produced a synergistic antitumor effect of combination therapy with PD-1 blockade and RFA against PLC.

The Activity of AQP9 Is Mediated MAPK in Arsenic-Treated Mouse Model of Hepatocellular Carcinoma

Background: Arsenic metabolism is primarily undergoing in hepatocytes, but the underlying mechanisms are to be defined. It is essential to study the response of aquaporin AQP9, protein kinase p38, and JNK with the stimulation of arsenic in mouse models of hepatocellular carcinoma. Methods: Mouse model of hepatocellular carcinoma (HCC) was induced with H22 cells injected subcutaneously on the lateral side of each right axilla in C57BL/6 mice. Then, western blotting and co-immunoprecipitation were used to detect the protein expression and phosphorylation of molecules AQP9, p38 and JNK in mouse models of hepatocellular carcinoma, respectively. The hepatocellular distribution of AQP9 was examined by the immunofluorescent method. Results: In both wild mice and a mouse model of liver tumor, there was no significant difference in the expressions of AQP9, protein kinase p38, and JNK after arsenic treatment, but the phosphorylation expression levels of the three were significantly increased to varying degrees, and the tumor model Compared with the wild-type group, the expression increased. Laser confocal experiments showed that in HepG2 cells, phosphorylated AQP9 was mainly distributed on the cell membrane under the stimulation of arsenic. Conclusion: Arsenic stimulation can increase the phosphorylation of AQP9, p38, and JNK in both wild-type C57 mice and liver tumor mice models. Arsenic stimulation facilitates phosphorylated-AQP9 predominantly distributed on the cell membrane of hepatoma cells HepG2.

Saponins of Marsdenia Tenacissima promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria then activating cytochrome C/Caspase-9/Caspase-3 pathway.

Liver cancer is one of the most common cancers in the world and the second leading cause of death in cancer patients. There is an urgent need for an effective and less toxic treatment for liver cancer. Saponins of Marsdenia Tenacissima (SMT) as a potential anticancer drug has attracted extensive attention of researchers because of its effective biological activity. The effect of SMT on HepG2 Li-7 and L-02 cells was detected by CCK8 assay. At the same time, the apoptosis rate was detected by flow cytometer and laser confocal microscope, and the morphological changes of mitochondria were observed under electron microscope. The levels of bax, cytochrome c, caspase-9, caspase-3, cleaved caspase-3 and protein were detected using Western bolt. Finally, BALB/c was subcutaneously injected with H22 cells to form tumors, and SMT was intragastrically injected to detect the size of the transplanted tumor. SMT can induce apoptosis in vitro and reduce the size of transplanted tumor in vivo. Increases the rate of apoptosis through the cytochrome c pathway and regulates the expression of apoptosis-related proteins. These results suggest that SMT may be one of the potential candidates for the treatment of liver cancer.

Protective effects of low-temperature plasma on cisplatin-induced nephrotoxicity

The application of atmospheric pressure low-temperature plasma (LTP) in medical treatment has received extensive attention owing to its redox regulatory and anti-inflammatory properties. Nephrotoxicity due to oxidative stress and inflammation is the main adverse effect of cisplatin. In the present study, rats with cisplatin-induced nephrotoxicity were treated with LTP to investigate its potential protective effect. The results showed that LTP treatment has multiple protective effects on cisplatin-induced nephrotoxicity. It significantly improved clinical indicators such as survival rate, water intake, food intake, body weight, and mobility, as well as physiological indexes such as reduced renal index and levels of serum urea, creatinine, and total bilirubin; pathological indicators such as reduced tubular injury, inflammatory infiltration, tubulointerstitial fibrosis, and apoptosis; cell survival indicators such as decreased protein levels of Caspase-3 and Bax and increased Bcl-2; anti-oxidation status such as reduced malondialdehyde content and increased activities of catalase, superoxide dismutase, and glutathione peroxidase; and reduced inflammatory factors such as TNF-α in kidney tissues. Specially, LTP treatment did not influence the anticancer effect of cisplatin as observed in the solid tumor mouse model established by subcutaneously inoculating H22 cells. Moreover, LTP did not influence the physiological and pathological indicators of normal rats, suggesting its biological safety. In conclusion, LTP can protect against cisplatin-induced nephrotoxicity through its anti-oxidation, anti-inflammation, and anti-apoptosis effects, without influencing the anticancer effect of cisplatin.

Ethanol extracts of Balanophora laxiflora Hemsl inhibit hepatocellular carcinoma with the involvement of HKII-mediated glycolysis

• UHPLC-Q-TOF-MS/MS was used to identify the main components of BLE. • BLE inhibits DEN-induced liver cancer in rats and damps lung metastasis in mice. • BLE inhibits HepG2 migration by regulating HKII-mediated aerobic glycolysis. Balanophora laxiflora Hemsl has been traditionally used to treat liver cancer. Eight ingredients were identified in the ethanol extracts of Balanophora laxiflora Hemsl (BLE) by UHPLC-Q-TOF-MS/MS analysis. BLE inhibited viability and migration of HepG2 and Hepa1-6 cells, but triggered negligible apoptosis. BLE also impeded glycolysis, glucose uptake and lactate generation. Hexokinase II (HKII), a key enzyme involved in glycolysis, was significantly repressed by BLE. HKII plasmid in HepG2 enhanced cell migration, glucose uptake and cell proliferation, which were all abrogated by BLE pre-treatment. BLE incubation also down-regulated the migration and glycolysis in HKII-overexpressed Hepa1-6 cells. The anti-cancer effect of BLE in vivo was evaluated. BLE inhibited the metastasis of H22 cells in lung and alleviated orthotopic liver cancer induced by diethylnitrosamine in rats. BLE also repressed tumor growth in mice bearing HepG2 cell xenografts. Therefore, BLE can be considered as a new drug candidate for the treatment of liver cancer.

Biejiajian Pill Promotes the Infiltration of CD8+ T Cells in Hepatocellular Carcinoma by Regulating the Expression of CCL5.

Tumor-infiltrating CD8+T lymphocytes are mostly associated with a favorable prognosis in numerous cancers, including hepatocellular carcinoma (HCC). Biejiajian Pill (BJJP) is a common type of traditional Chinese medicine that is widely used in the treatment of HCC in China. Previous studies showed that BJJP suppressed the growth of HCC cells both in vivo and in vitro, by exerting direct cytotoxic effects on tumor cells. The present study demonstrated that in addition to direct cytotoxicity, BJJP inhibits the growth of tumor cells by promoting the infiltration of CD8+T cells into the tumor in H22-bearing mice. Mechanistically, chemokine ligand 5 (CCL5) was identified as one of the most highly expressed chemokines by tumor cells in vivo after treatment with BJJP. Additionally, CCL5 was knocked down in H22 cells and the results showed that knockdown of the gene significantly impaired the infiltration of CD8+T cells in vivo. Furthermore, the effects of BJJP on human HCC cell lines were assessed in vitro. Similarly, cells treated with BJJP had higher expression of CCL5 mRNA, which was consistent with increased levels of CCL5 protein in human tumor cells. These findings provide new insights into the anticancer effects of BJJP, which regulated the expression of CCL5 and the infiltration of CD8+T cells. The results, therefore, suggest that BJJP has great potential application in clinical practice.

Anti-Tumor Effects of Carrimycin and Monomeric Isovalerylspiramycin I on Hepatocellular Carcinoma in Vitro and in Vivo.

Hepatocellular carcinoma results in a high risk of second primary malignancies and has prominent morbidity and mortality. There is a lack of effective treatment and prognosis is poor. Therefore, effective drugs need to be discovered. Carrimycin is a 16-member macrolide antibiotic with anticancer activity, and monomeric isovalerylspiramycin I is a main component. The aim of this study was to determine the anti-tumor effects of carrimycin and monomeric isovalerylspiramycin I on hepatocellular carcinoma through in vivo and in vitro experiments. In vitro, changes in cellular proliferation, migration, invasion, and apoptosis were analyzed by MTT, colony formation, EdU labeling, wound-healing, matrigel transwell invasion, and flow cytometric assays using SK-Hep1, Hep3B, SNU-354, SNU-387 hepatocellular carcinoma cell lines. Western blotting and RT-PCR were used to detect the effects of carrimycin and monomeric isovalerylspiramycin I on the expression levels of vascular endothelial growth factor (VEGF) and programmed death ligand 1 (PD-L1). Nude mice were subcutaneously transplanted with SK-Hep1 cells or C57BL/6J mice were orthotopically transplanted with hepatocarcinoma H22 cells. Tumor volume, pathological changes in tumor tissues, and the concentration of VEGF in mouse serum were measured after treatments. Carrimycin and monomeric isovalerylspiramycin I dose-dependently inhibited hepatocellular carcinoma cell viability, colony formation, and DNA replication. These agents markedly suppressed migration and invasion and promoted apoptosis of the cell lines. Western blotting and RT-PCR demonstrated that carrimycin and monomeric isovalerylspiramycin I reduced VEGF and PD-L1 protein and mRNA levels in a dose-dependent manner. In vivo studies further confirmed that carrimycin and monomeric isovalerylspiramycin I could significantly inhibit tumor growth, tumor histopathological alterations, and the concentration of VEGF in both mouse tumor models. These results show that carrimycin and monomeric isovalerylspiramycin I promoted apoptosis and inhibited proliferation, migration, and invasion of hepatocellular carcinoma cells. Therefore, our discovery suggests anti-tumor capacity for carrimycin and monomeric isovalerylspiramycin I and provides data on potential new drugs for inhibiting hepatocellular carcinoma.

Excretory-secretory product of Trichinella spiralis inhibits tumor cell growth by regulating the immune response and inducing apoptosis

The excretory-secretory product (ESP) of Trichinella spiralis (T. spiralis) has been reported to inhibit the growth of various tumor cells, but the mechanism is not yet clear. To explore the effect and mechanism of ESP on liver cancer cells, tumor models were established with H22 cells and then infected with T. spiralis. The results showed that the growth of tumors in mice infected with T. spiralis was significantly inhibited. ESP from adult worms or muscle larvae were then incubated with H22 cells in vitro, and it was found that the ESP could inhibit cell proliferation and promote apoptosis. Subsequently, apoptosis-related proteins in stimulated H22 cells were evaluated, and ESP was found to induce cell apoptosis through the mitochondrial pathway. Additionally, Th-related cytokines were investigated in vivo, and the results showed that the levels of Th1 cytokines were significantly increased in the early stage of T. spiralis infection, while Th2 cytokines increased later than Th1 cytokines, implying that Th1 cytokines with antitumor effects may play a role in inhibiting tumor growth at early stage. In short, ESP can directly induce tumor cell apoptosis and indirectly inhibit tumor cell growth through the host immune system, which may be the antitumor mechanism of T. spiralis infection.

Marchantia polymorpha L. ethanol extract induces apoptosis in hepatocellular carcinoma cells via intrinsic- and endoplasmic reticulum stress-associated pathways

BackgroundMarchantia polymorpha L. is a kind of Chinese herbal medicine and has various biological activities including antioxidant and antifungal. However, it is not clear about the antitumor effect and mechanism of M. polymorpha. We prepared M. polymorpha ethanol extract (MPEE) and investigated its antitumor effect on hepatocellular carcinoma cells both in vitro and in vivo.MethodsThe viability of hepatocellular carcinoma cells was detected by MTT assay. The distribution of cell cycle was analyzed by propidium iodide (PI) staining. The morphology of nuclei was observed by Hoechst 33258 staining. Apoptosis was detected by Annexin V/PI staining. JC-1 fluorescent probe and DCFH-DA were used to detect the mitochondrial membrane potential (ΔψM) and the level of reactive oxygen species (ROS), respectively. Caspase inhibitors were used to test the function of caspase in the induction of apoptosis. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the levels of mRNA and protein, respectively. Differentially expressed genes and signaling pathways were identified by transcriptome analysis. The H22 tumor mouse model was used to detect the antitumor effect of the extract.ResultsMPEE significantly suppressed the migration and growth of BEL-7404, HepG2 and H22 cells in a dose- and time-dependent manner through induction of apoptosis characterized by chromosomal condensation and cell cycle arrest at G0/G1 and G2/M phases. MPEE induced mitochondria-dependent apoptosis via upregulation of Bax and downregulation of Bcl-2 to reduce mitochondrial membrane potential and increase the release of cytochrome c. The levels of cleaved caspase-8 and -9 were significantly increased, which sequentially activated caspase-3 to cleave PARP. We further found that MPEE significantly increased ROS production and activated endoplasmic reticulum (ER) stress associated-apoptotic signaling pathway. Moreover, MPEE significantly inhibited H22 tumor growth in mouse model and improved the survival of tumor mice.ConclusionThese results suggested that MPEE suppressed hepatocellular carcinoma cell growth through induction of apoptosis via intrinsic- and ER stress-associated pathways.

Gut microbiota enhances the chemosensitivity of hepatocellular carcinoma to 5-fluorouracil in vivo by increasing curcumin bioavailability.

5-Fluorouracil (5-Fu) is efficient for hepatocellular carcinoma (HCC) treatment, but fast-emerging resistance limits its usage. Curcumin is being investigated for its potential chemosensitivity, but its low oral bioavailability hinders its chemosensitivity effect in vivo. Gut microbiota modulation is considered to contribute to its bioactivities in vivo. In the current study, we demonstrate that curcumin can enhance 5-Fu chemosensitivity in HCC cells in vitro, increase the apoptosis rate, arrest the cell cycle at G2/M phase, and block the PI3k/AKT/mTOR signalling pathway by inhibiting the phosphorylation of PI3K and its downstream protein kinases. Curcumin also remarkably sensitized H22 cells to 5-Fu, allowing it to inhibit tumour growth in vivo. 16S rDNA sequencing suggests that curcumin in combination with 5-Fu significantly alters the gut microbiota composition based on alpha and beta diversity analysis compared to drug treatment alone. Gut microbiota depletion abolished curcumin's chemosensitivity effect in vivo. A pharmacodynamics study suggested that the gut microbiota increased the oral bioavailability of curcumin (AUC(0-t) 15.24 ± 0.77 μM/h [wt] vs. 3.04 ± 0.18 μM/h [gut microbiota depleted]). In conclusion, curcumin can increase the chemosensitivity of HCC to 5-Fu in vitro and in vivo, and gut microbiota plays a key role in its effect in vivo.

MiR‑29b suppresses proliferation and induces apoptosis of hepatocellular carcinoma ascites H22 cells viaregulating TGF‑β1 and p53 signaling pathway.

MicroRNA (miR)-29b is a key tumor regulator. It can inhibit tumor cell proliferation, induce apoptosis, suppress tumor invasion and migration, thus delaying tumor progression. Our previous studies revealed an increased level of miR-29b in hepatoma 22 (H22) cells in ascites tumor-bearing mice. The present study investigated the effect of miR-29b on proliferation and apoptosis of hepatocellular carcinoma ascites H22 cells and its association with the transforming growth factor-β1 (TGF-β1) signaling pathway and p53-mediated apoptotic pathway. Briefly, H22 cells were transfected with miR-29b-3p (hereinafter referred to as miR-29b) mimic or miR-29b inhibitor. MTS cell proliferation assay and flow cytometry were used to analyze cell viability and apoptosis. The expression change of the TGF-β1 signaling pathway and p53-mediated apoptotic pathway were detected by reverse transcription-quantitative PCR, western blotting and immunofluorescence. Furthermore, cells were treated with exogenous TGF-β1 and TGF-β1 small interfering RNA to evaluate the crosstalk between TGF-β1 and p53 under miR-29b regulation. The overexpression of miR-29b decreased cell viability, increased cell apoptosis, activated the TGF-β1 signaling pathway and p53-mediated apoptotic pathway. Conversely, these effects were reversed by the miR-29b inhibitor. Moreover, the effect of miR-29b mimic was further increased after treating cells with exogenous TGF-β1. The activation of the TGF-β1 signaling pathway and p53-mediated apoptotic pathway induced by miR-29b overexpression were reversed by TGF-β1 inhibition. In summary, these data indicated that miR-29b has an important role in proliferation and apoptosis of H22 cells by regulating the TGF-β1 signaling pathway, the p53-dependent apoptotic pathway, and the crosstalk between TGF-β1 and p53.

Nrf2 Down-Regulation by Camptothecin Favors Inhibiting Invasion, Metastasis and Angiogenesis in Hepatocellular Carcinoma.

Higher oxidant stress capacity could promote invasion and metastasis. A previous study showed hepatocellular carcinoma (HCC) expressed more Nrf2 than para-carcinoma tissue. The chemotherapeutics such as epirubicin (EPI) could increase Nrf2 expression, while Camptothecin (CPT) could inhibit tumor growth by down-regulating the key molecule of antioxidant stress signal—Nrf2. The role of Nrf2 in invasion and metastasis was still unclear. In this study, we use EPI and CPT to determine the invasion and metastasis in Huh7 cells, H22 and Huh7 mouse models. In Huh7 cells, Nrf2 expression and ROS level were found increased after incubation with EPI by western blot and flow cytometry assay. But with the combination of EPI and CPT, inhibition of Nrf2 could decrease proliferation, invasion, and metastasis, which were investigated by CCK8 assay, wound healing, and Transwell assays. In Huh7 and H22 mouse models, EPI promoted Nrf2 up-regulation and nucleus translocation. Tumor growth was obviously inhibited with a single application of EPI or CPT. The combination of EPI and CPT could inhibit Nrf2 expression but demonstrated more suppressing effect of tumor growth than EPI. Western blot and immunohistochemical staining study revealed that Nrf2 inhibition was beneficial in decreasing the expression of N-cadherin, MMP9, Snail as well as Twist, and increasing E-cadherin, which were associated with epithelial–mesenchymal transition (EMT). Nrf2 down-regulation promoted lung metastasis of H22 cells in vivo. In addition, H&E staining and immunofluorescence staining of VEGFR suggested angiogenesis of Huh7 and H22 tumors was reduced. In conclusion, down-regulation of Nrf2 demonstrated inhibition of invasion, metastasis, and angiogenesis of hepatoma, which may provide a potential therapy in HCC.

Nanobubbles Containing sPD-1 and Ce6 Mediate Combination Immunotherapy and Suppress Hepatocellular Carcinoma in Mice.

PurposeImmune checkpoint inhibitors (ICIs) and sonodynamic therapy (SDT) are types of immunotherapy. In order to combine soluble programmed cell death protein 1 (sPD-1)-mediated immune checkpoint therapy and chlorin e6 (Ce6)-assisted SDT, nanobubbles (NBs) were generated to simultaneously load sPD-1 and Ce6.Materials and MethodsThe sPD-1/Ce6-NBs, which were prepared by thin-film hydration and mechanical oscillation, had a stable physical condition, and delivered sPD-1 and Ce6 in a targeted manner. NBs could strengthen tumor suppression by increasing tumor-targeting accumulation of Ce6 and sPD-1, and by inducing ultrasound-targeted NB destruction. A mouse H22 cell hepatoma xenograft model was used to evaluate the synergetic immunotherapeutic effect and mechanism of sPD-1/Ce6-NBs.ResultsBy observing the tumor inhibition rate, tissue and cell apoptosis, apoptosis-related genes and protein expression, the best immunotherapeutic effect was exhibited by the sPD-1/Ce6-NBs group. The immunotherapeutic mechanism initially demonstrated that when tumor cells were transfected by sPD-1 delivered by NBs, which downregulated the expression of programmed death-ligand 1 (PD-L1) in tumor cells, and blocked the PD-1/PD-L1 signaling pathway, which improved T-cell-mediated tumor inhibition. Furthermore, ICIs combined with SDT induced immunogenic cell death by translocating calreticulin to the cell surface and then synergistically enhancing antitumor immune responses.ConclusionIn conclusion, sPD-1/Ce6-NBs were successfully designed. Ultrasound-mediated sPD-1/Ce6-NBs are potentially effective delivery systems for combination immunotherapy of hepatocellular carcinoma.

The Structural Characterization of a Novel Water-Soluble Polysaccharide from Edible Mushroom Leucopaxillus giganteus and Its Antitumor Activity on H22 Tumor-Bearing Mice.

In the present study, a novel cold water-soluble polysaccharide fraction (LGP) with the average molecular weight of 1.78×106 Da was extracted and purified from Leucopaxillus giganteus and its primary structure as well as in vivo antitumor activity was evaluated. The monosaccharide composition of LGP was determined by ion chromatography to be galactose, xylose, glucose and fucose in a molar ratio of 2.568 : 1.209 : 1 : 0.853. Its backbone was composed of α-D-Glu, α-D-Xyl, α-D-Gal and α-L-Fuc. The results of in vivo antitumor experiment demonstrated that LGP could effectively protect immune organs, has excellent antitumor activity, and inhibit the proliferation of H22 solid tumors in a dose-dependent manner. By analyzing Annexin V-FITC/PI staining, cell cycle and mitochondrial membrane potential detection assay, we concluded that LGP induced apoptosis of H22 cells via S phase arrest and mitochondria-mediated apoptotic pathway. Our results could provide valuable information for the potential application of LGP as an anti-hepatoma agent.

The Collective Effect of MIP-3α and FL Promotes Dendritic Cell Function Within the Immune Microenvironment of Murine Liver Cancer.

Hepatocellular carcinoma is a highly malignant and lethal tumor. In addition to surgery, immunotherapy is currently a more effective treatment for hepatocellular carcinoma. The tumor immune microenvironment (TIME) largely determines the efficacy of cancer immunotherapy. Based on the universal targeting of TIME modulators in clinical treatment, TIME modulators are promising targets for tumor immunotherapy. We investigated the effect of a double gene expression vector (recombinant galactose-terminal glycol-poly-L-lysine coupled MIP-3α-FL) on dendritic cells (DCs) regulation within the TIME of mice with liver cancer. H22 cells were transfected with a recombinant MIP-3α-FL plasmid to induce DCs differentiation and chemotaxis. The effects of transfection were investigated by flow cytometry following the modified Boyden’s method. Cytokine-induced killer (CIK) cells co-culture revealed changes in the antigen presentation ability of DCs. Further, tumor-bearing mice were injected with the recombinant double gene vector via the tail vein. We compared the survival time, tumor volume, weight of the mice, as well as the number and phenotype of tumor-infiltrating DCs (TIDCs) between groups. The supernatant of transfected H22 cells promoted the phenotypic maturation of DCs, enhancing their chemotaxis. Further, treated DCs promoted the cytokine secretion and killing ability of CIK cells. The survival time of mice injected with the double gene vector was significantly prolonged, while their tumor weight and volume were relatively reduced. Flow cytometry revealed that the number of TIDCs (as well as CD80 and CD86 expression) in the MouseMIP-3α-FL group, were significantly higher than in the control group. The combination of MIP-3α and FL can significantly promote DCs aggregation, maturation, and enhance their antigen presentation ability. The coupling of the double gene vector with glycosylated polylysine can improve the precise targeting of the liver and inhibit tumor growth in vivo, providing a novel approach for immune therapy in liver cancer.