Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1ß, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1ß mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1ß, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1ß after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

Network pharmacology combined with experimental validation to investigate the effect of Rongjin Niantong Fang on chondrocyte apoptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a chronic degenerative disease that affects the quality of life of middle­aged and elderly individuals, and is one of the major factors leading to disability. Rongjin Niantong Fang (RJNTF) can alleviate the clinical symptoms of patients with KOA, but the molecular mechanism underlying its beneficial effects on KOA remains unknown. Using pharmacological analysis and in vitro experiments, the active components of RJNTF were analyzed to explore their potential therapeutic targets and mechanisms in KOA. The potential targets and core signaling pathways by which RJNTF exerts its effects on KOA were obtained from databases such as Gene Expression Omnibus, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform. Subsequently, chondrocyte apoptosis was modeled using hydrogen peroxide (H2O2). Cell Counting Kit­8 assay involving a poly [ADP­ribose] polymerase­1 (PARP1) inhibitor, DAPI staining, reverse transcription­quantitative PCR, Annexin V­FITC/PI staining and flow cytometry, western blotting and co­immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1­knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H2O2­induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase­3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis­inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase­3, PARP1 total protein, as well as the cytoplasmic expression of AIF and MIF, suggesting that RJNTF may inhibit chondrocyte apoptosis through the PARP1/AIF signaling pathway.

Low serum apolipoprotein A1 level predicts poor prognosis of patients with diffuse large B-cell lymphoma in the real world: a retrospective study.

BACKGROUND: Apolipoprotein A1 (ApoA1) is a member of the apolipoprotein family with diverse functions. It is associated with the pathogenesis and prognosis of several types of tumors. However, the role of serum apolipoprotein A1 (ApoA1) in the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) remains unclear. This study aimed to elucidate its influence on clinical outcomes in patients with DLBCL. METHODS: We retrospectively analyzed a cohort of 1583 consecutive DLBCL patients admitted to the Fujian Medical University Union Hospital between January 2011 and December 2021. 949 newly diagnosed DLBCL patients who met the inclusion criteria were enrolled for statistical analysis. Receiver operating characteristic curve analysis was performed to determine the optimal cut-off value for serum ApoA1 levels for prognostic prediction among patients with DLBCL. The correlations between serum ApoA1 levels and clinical and laboratory parameters were analyzed. Prognostic significance was analyzed using univariate and multivariate Cox proportional hazards models. RESULTS: Newly diagnosed patients with DLBCL demonstrated low serum ApoA1 levels (< 0.925 g/L), had more B symptoms, higher levels of serum lactate dehydrogenase (LDH) (>upper limit of normal), poorer performance status (Eastern Cooperative Oncology Group score of 2-4), higher percentage of advanced stage and non-germinal center B-cell (non-GCB) subtype, more cases of > 1 extranodal site, higher International Prognostic Index (IPI) score (3-5), and higher incidence of relapse or refractory diseases compared with those with high serum ApoA1 levels (≥ 0.925 g/L). Low serum ApoA1 levels were an independent adverse prognostic factor for overall survival (OS) but not progression-free survival (PFS). CONCLUSIONS: Low serum ApoA1 levels were associated with poor treatment response and inferior survival in newly diagnosed patients with DLBCL.

Melatonin enhances anti-tumor immunity by targeting macrophages PD-L1 via exosomes derived from gastric cancer cells.

Melatonin (MLT) is a hormone with potential anti-tumor properties, but the molecular mechanisms remain unclear. The present study aimed to explore the effect of MLT on exosomes derived from gastric cancer cells, with the goal of gaining insight into its anti-tumor activity. Results from in vitro experiments showed that MLT was able to enhance the anti-tumor activity of macrophages that had been suppressed by exosomes from gastric cancer cells. This effect was achieved through regulation of the levels of PD-L1 in macrophages via modulation of the associated microRNAs in the cancer-derived exosomes. Furthermore, MLT treatment increased the secretion of TNF-α and CXCL10 by the macrophages. Besides, MLT treatment of gastric cancer cells led to the production of exosomes that promoted the recruitment of CD8+ T cells to the tumor site, resulting in inhibition of tumor growth. Collectively, these results provide evidence for the modulation of the tumor immune microenvironment by MLT through regulation of exosomes derived from gastric cancer cells, suggesting a potential role for MLT in novel anti-tumor immunotherapies.

Bufalin Inhibits Tumorigenesis, Stemness, and Epithelial-Mesenchymal Transition in Colorectal Cancer through a C-Kit/Slug Signaling Axis.

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin's function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of ß-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial-mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin's anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.

Dendrobium mixture improves gestational diabetes mellitus through regulating Nrf2/HO1 signaling pathway.

BACKGROUND: Gestational diabetes mellitus (GDM) is characterized by insulin resistance during pregnancy, and it is always combined with serious complications. Dendrobium mixture (DMix) is a kind of traditional Chinese medicine, and it has been proved to be an effective treatment for diabetes. However, the regulatory role of DMix in GDM remains elusive. METHODS: High fat feed combined with streptozotocin injection and high glucose medium were used to establish GDM animal and cell models, respectively. The levels of blood glucose, blood lipid, and insulin were measured with commercial kits. Western blotting was used to detect protein expression. RESULTS: DMix improved pancreas and placenta injury in GDM rats. DMix reversed the influence of GDM on the levels of SOD, MDA, and glutathione in the serum. Hyperglycemia and hyperlipidemia in GDM rats were suppressed by DMix. The activation of MAPK and inhibition of Nrf2/HO1 in GDM animal and cell models were reversed by DMix. The increase of ROS intensity, apoptosis, and inflammation factors in HG treated cells were reversed by DMix. CONCLUSION: This research proved that DMix improved GDM through inhibiting oxidative condition, inflammation factors, hyperglycemia and hyperlipidemia. This study might provide a novel thought for the prevention and treatment of GDM.

Screening and Validation of Significant Genes with Poor Prognosis in Pathologic Stage-I Lung Adenocarcinoma.

Background: Although more pathologic stage-I lung adenocarcinoma (LUAD) was diagnosed recently, some relapsed or distantly metastasized shortly after radical resection. The study aimed to identify biomarkers predicting prognosis in the pathologic stage-I LUAD and improve the understanding of the mechanisms involved in tumorigenesis. Methods: We obtained the expression profiling data for non-small cell lung cancer (NSCLC) patients from the NCBI-GEO database. Differentially expressed genes (DEGs) between early-stage NSCLC and normal lung tissue were determined. After function enrichment analyses on DEGs, the protein-protein interaction (PPI) network was built and analyzed with the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape. Overall survival (OS) and mRNA levels of genes were performed with Kaplan-Meier analysis and Gene Expression Profiling Interactive Analysis (GEPIA). qPCR and western blot analysis of hub genes in stage-I LUAD patients validated the significant genes with poor prognosis. Results: A total of 172 DEGs were identified, which were mainly enriched in terms related to management of extracellular matrix (ECM), receptor signaling pathway, cell adhesion, activity of endopeptidase, and receptor. The PPI network identified 11 upregulated hub genes that were significantly associated with OS in NSCLC and highly expressed in NSCLC tissues compared with normal tissues by GEPIA. Elevated expression of ANLN, EXO1, KIAA0101, RRM2, TOP2A, and UBE2T were identified as potential risk factors in pathologic stage-I LUAD. Except for ANLN and KIAA0101, the hub genes mRNA levels were higher in tumors compared with adjacent non-cancerous samples in the qPCR analysis. The hub genes protein levels were also overexpressed in tumors. In vitro experiments showed that knockdown of UBE2T in LUAD cell lines could inhibit cell proliferation and cycle progression. Conclusions: The DEGs can probably be used as potential predictors for stage-I LUAD worse prognosis and UBE2T may be a potential tumor promoter and target for treatment.

Inhibition of DCLK1 with DCLK1-IN-1 Suppresses Renal Cell Carcinoma Invasion and Stemness and Promotes Cytotoxic T-Cell-Mediated Anti-Tumor Immunity.

The approval of immune checkpoint inhibitors has expanded treatment options for renal cell carcinoma (RCC), but new therapies that target RCC stemness and promote anti-tumor immunity are needed. Previous findings demonstrate that doublecortin-like kinase 1 (DCLK1) regulates stemness and is associated with RCC disease progression. Herein, we demonstrate that small-molecule kinase inhibitor DCLK1-IN-1 strongly inhibits DCLK1 phosphorylation and downregulates pluripotency factors and cancer stem cell (CSC) or epithelial-mesenchymal transition (EMT)-associated markers including c-MET, c-MYC, and N-Cadherin in RCC cell lines. Functionally, DCLK1-IN-1 treatment resulted in significantly reduced colony formation, migration, and invasion. Additionally, assays using floating or Matrigel spheroid protocols demonstrated potent inhibition of stemness. An analysis of clinical populations showed that DCLK1 predicts RCC survival and that its expression is correlated with reduced CD8+ cytotoxic T-cell infiltration and increases in M2 immunosuppressive macrophage populations. The treatment of RCC cells with DCLK1-IN-1 significantly reduced the expression of immune checkpoint ligand PD-L1, and co-culture assays using peripheral blood monocytes (PBMCs) or T-cell expanded PBMCs demonstrated a significant increase in immune-mediated cytotoxicity alone or in combination with anti-PD1 therapy. Together, these findings demonstrate broad susceptibility to DCLK1 kinase inhibition in RCC using DCLK1-IN-1 and provide the first direct evidence for DCLK1-IN-1 as an immuno-oncology agent.

Masking quercetin: A simple strategy for selective detection of rutin by combination of bovine serum albumin and fluorescent silicon nanoparticles.

As a typical kind of bioactive flavonoid glycoside, rutin and its aglycone quercetin possess similar chemical structures and properties. It still remains a challenge to achieve reliably and accurately detection of rutin in the presence of quercetin. In this work, a simple fluorescent method combining water-dispersed silicon nanoparticles (SiNPs) with bovine serum albumin (BSA) were constructed for the selective detection of rutin in the presence of quercetin and other common compounds in traditional Chinese herbs. SiNPs with high fluorescent quantum yield and good thermostability were prepared by one-pot hydrothermal method using ferulic acid as the reduction reagent for the first time. The fluorescence of SiNPs could be obviously quenched both by rutin and quercetin in phosphate buffer solution. Interestingly, when the solution contained certain concentration of BSA, the fluorescence of the SiNPs can only be remarkably quenched by rutin. The innovative use of BSA to block the interference of quercetin make it possible to selectively detect of rutin by fluorescence spectrometry under the coexistence of quercetin. Under the optimum conditions, the fluorescence displayed a linear decrease response as the rutin concentration increased in the range of 0.33-33.30 µM with a detection limit of 0.04 µM (S/N = 3). The possible quenching mechanism of rutin to SiNPs has also explored and concluded to be mainly caused by inner filter effect. This work provides a novel methodology for the simple, low-cost and selective determination method for rutin.

Qingjie Fuzheng Granules regulates cancer cell proliferation, apoptosis and tumor angiogenesis in colorectal cancer xenograft mice via Sonic Hedgehog pathway.

BACKGROUND: Sonic Hedgehog (SHh) signaling pathway plays a critical role in cell proliferation, apoptosis, and tumor angiogenesis in various types of malignancies including colorectal cancer (CRC). Qingjie Fuzheng Granules (QFG) is a traditional Chinese medicinal formula, which has been clinically used in various cancer treatments, including CRC. In this study, we explored the potential molecular mechanisms of QFG treatment effects on CRC via the SHh pathway. METHODS: A CRC HCT-116 xenograft mouse model was utilized for all experiments. Mice were treated with intra-gastric administration of 1 g/kg of QFG or saline 6 days a week for 28 days (4 weeks). Body weight, length and shortest diameter of the tumor were measured every 3 days. At the end of the treatment, the tumor weight was measured. TUNEL staining assays were used to detect tumor apoptosis. Western blot and immunohistochemistry (IHC) assays were used to detect the expression of relative proteins. RESULTS: In our results, QFG inhibited the increase of tumor volume and weight, and exhibited no impact on mouse body weight. Furthermore, QFG significantly decreased the expression of SHh, Smo and Gli proteins, indicating the action of SHh signaling. Consequently, the expression of pro-proliferative survivin, Ki-67, Cyclin-D1 and CDK4 were decreased and expression of anti-proliferative p21 was increased. The pro-apoptotic Bax/Bcl-2 ratio, cle-caspase-3 and TUNEL-positive cell percentage in tumor tissues were increased. Meanwhile, the pro-angiogenic VEGF-A and VEGFR-2 expression was down-regulated. CONCLUSIONS: QFG inhibited CRC cell proliferation and promoted CRC cell apoptosis and tumor angiogenesis in vivo through the suppression of SHh pathway, suggesting that QFG could be a potential therapeutic drug for CRC.

A nanometer-sized protease inhibitor for precise cancer diagnosis and treatment.

Inhibition of pro-cancer proteases is a potent anticancer strategy. However, protease inhibitors are mostly developed in the forms of small molecules or peptides, which normally suffer from insufficient metabolic stability. The fast clearance significantly impairs the antitumor effects of these inhibitors. In this study, we report a nanometer-sized inhibitor of a pro-cancer protease, suppressor of tumorigenicity 14 (st14), which has been reported as a potent prognostic marker for multiple cancers. This st14 inhibitor was fabricated by conjugating a recombinant st14 inhibitor (KD1) with carbon quantum dots (CQDs). CQD-KD1 not only demonstrated high potency of inhibiting st14 activity in biochemical experiments, but also remarkably suppressed the invasion of breast cancer cells. In contrast to the original recombinant KD1, CQD-KD1 demonstrated a prolonged retention time in plasma and at the tumor site because of the reduced renal clearance. Consistently, CQD-KD1 demonstrated enhanced efficacies of suppressing tumor growth and cancer metastases in vivo. In addition, CQD-KD1 precisely imaged tumor tissues in cancer-grafted mice by specifically targeting the over-expressed st14 on the tumor cell surface, which indicates CQD-KD1 as a potent probe for the fluorescence guided surgery of tumor resection. In conclusion, this study demonstrates that CQD-KD1 is a highly potent diagnostic and therapeutic agent for cancer treatments.

Effect of melatonin on T/B cell activation and immune regulation in pinealectomy mice.

Melatonin is an indole neuroendocrine hormone that is mainly secreted by the pineal gland to regulate circadian rhythm, antioxidation, and immune regulation. Melatonin plays an important role in T cell-mediated immune responses against cancer, infections, and the development of many autoimmune diseases. The aim of this study was to investigate the immunomodulatory effects of melatonin on T/B cell activation in pinealectomy mice. The improved pinealectomy procedure for mice presented in this study is a good animal model to be used in follow-up studies on melatonin. After pinealectomy, the tissue removed was identified as the pineal body using HE staining. The effects of melatonin supplementation on T cell activation and activation-related changes to the MAPK/NF-κ B pathways were analyzed by flow cytometry and real-time PCR. We found that expression levels of Th1, Th2 and Th17-related cytokines in peripheral blood were lower in mice that had undergone pinealectomy, compared with normal mice. After melatonin supplementation, cytokine levels rapidly increased within a short period of time, which resulted in the gradual recovery of cytokine expression levels. Moreover, activation of T/B cells in mice was weakened and decreased after pineal gland removal. Melatonin was found to inhibit the expression of TLR3, p38, JNK, and MAPK/NF-κ B within a short period (2 weeks) of melatonin replenishment. This inhibition gradually weakened with time, since the degree of inhibition is negatively related with the dosage of melatonin. In conclusion, melatonin may regulate the activation of T/B cells, playing a critical role in the regulation of immune balance.

Activation of the Toll­like receptor 2 signaling pathway inhibits the proliferation of HCC cells in vitro.

Toll­like receptor 2 (TLR2), is an important pattern recognition receptor which serves a role in chronic inflammation of the liver. However, the role of TLR2 in the progression of human hepatocellular carcinoma (HCC) remains unknown. The aim of the present study was to examine the effects of the activation of the TLR2 signaling pathway on biological functions, such as proliferation and apoptosis. TLR2 expression in HCC tissues was assayed by quantitative polymerase chain reaction, flow cytometry and western blotting. B76/Huh7 cells were transfected with overexpression plasmids, and cell proliferation was detected using a Cell Counting Kit­8 assay and the secreted cytokines in the supernatant of transfected cells were measured by ELISA. The findings revealed that TLR2 expression was increased in the peritumoral groups compared with inner­tumoral groups. Activation of the TLR2 signaling pathway through overexpression of pathway molecules inhibited the growth of B76/Huh7 cells and the secretion of interleukin­6 and tumor necrosis factor­α were reduced. Inhibition of the TLR2 signaling pathway resulted in a significant increase in the downstream signaling cascade, thus potentially increasing hepatocarcinogenesis and tumor progression. Activation of the TLR2 signaling pathway may be a potential target for therapeutic intervention in patients with HCC and downstream secreted cytokines are required for the functional biological effect. Therefore, modulation of the TLR2 signaling pathway may provide important insight into designing effective therapeutic regimens for treating patients with HCC.

3, 3'-Dimethylquercetin Inhibits the Proliferation of Human Colon Cancer RKO Cells through Inducing G2/M Cell Cycle Arrest and Apoptosis.

BACKGROUND: Our previous study successfully identified that 3,3'-Dimethylquercetin (DMQ) acted as a potent anticancer agent against human colon cancer cell lines RKO. Thus, this study was conducted to investigate the underlying mechanism by which DMQ displayed inhibitory activity in RKO cells. METHODS: Flow cytometry was used to evaluate the effect of DMQ on the cell cycle arrest, as well as the mitochondrial membrane potential in RKO cells. DAPI staining and DNA fragmentation ladder assays were performed to assess the apoptosis inducing activity of DMQ. Furthermore, western blot analysis was conducted to examine the expression of related proteins responsible for the cell cycle arrest and apoptosis. RESULTS: Treatment with DMQ caused a significant increase in the fraction of G2/M cells, and induced remarkable apoptosis. Furthermore, western blot analysis showed that DMQ arrested cells at G2/M checkpoint by down-regulation of cyclin B1, cdc2 and cdc25c and up-regulation of p21, and induced cell apoptosis via affecting the ratio of Bax/Bcl-2, causing loss of the mitochondrial membrane potential and enhancing the expression of cleaved caspase-9 (C-caspase-9) and cleaved caspase-3 (C-caspase-3). CONCLUSION: These data showed that DMQ could suppress RKO cell growth by arresting RKO cells at G2/M checkpoint and inducing mitochondria-dependent cell apoptosis. Our findings shed light on the potential use of DMQ as a chemotherapeutic agent for CRC.

Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice.

Fuzheng Qingjie (FZQJ) is a polyherbal Chinese medicine that has previously been implemented as an adjuvant therapy for gastrointestinal cancer. The present study investigated whether FZQJ is able to potentiate the anticancer effect of cyclophosphamide (CTX). Hepatoma 22 tumor-bearing mice were randomly divided into a vehicle group, CTX group, FZQJ group and combination (CTX+FZQJ) group. In addition, untreated mice without H22 cells served as blank controls. Seven days post-treatment, the mice were sacrificed and the tumors were weighed. Blood cells were evaluated using an automatic hemocytometer analyzer and flow cytometer. The expression levels of interleukin (IL)-2 and tumor necrosis factor (TNF)-α were evaluated using a radioimmunoassay. Apoptotic cells were observed using a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Alanine transaminase, aspartate aminotransferase, blood urea nitrogen and creatinine were examined using an automatic biochemical analyzer. The results demonstrated that the tumor inhibitory rate and apoptosis index were higher in the combination group, compared with those in the CTX group. Notably, FZQJ was able to alleviate CTX-induced decreases in the numbers of white blood cells and platelets, CD3+ and CD4+ T lymphocyte subsets, and the concentration of hemoglobin, body weight and thymus index, and increase serum TNF-α and IL-2 levels without overt hepatorenal toxicity. These results suggest that FZQJ granules may enhance the anticancer effect of CTX, in addition to alleviating the side effects.

Fuzheng Qingjie Granules Inhibit Growth of Hepatoma Cells via Inducing Mitochondria-Mediated Apoptosis and Enhancing Immune Function.

Fuzheng Qingjie (FZQJ) granules, a compound Chinese medicine, have been used as an adjuvant therapy for alimentary tract cancers. However, the underlying anticancer mechanisms are still not well understood. In the present study, HepG2 cells were treated with FZQJ-containing serum. Cell proliferation was evaluated using MTT assay. Apoptosis was analyzed using a flow cytometer. Cell ultrastructure was observed under a transmission electron microscope. The mitochondrial membrane potential (Δψ) was examined with JC-1 dye. In H22 tumor-bearing mice, CD4+ T cells, CD8+ T cells, CD3+ T cells, and natural killer (NK) cells in peripheral blood were evaluated cytometrically. Interleukin (IL)-2 and tumor necrosis factor (TNF)-α levels were measured using radioimmunoassay.The mRNA levels of Bax and Bcl-2 were examined by reverse transcription-polymerase chain reaction. The protein levels of Bax, Bcl-2, cytochrome C, caspase 3 and 9, PARP, and CD69 were examined by Western blotting. The apoptotic cells in tissues were observed using TUNEL method. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were detected by an automatic biochemical analyzer. The results showed that FZQJ-containing serum remarkably inhibited proliferation of HepG2 cells in dose- and time-dependent manners, induced HepG2 cell apoptosis and caused a decrease of Δψ. Analysis of tumor tissue showed that FZQJ-induced apoptosis was accompanied by downregulation of Bcl-2 and upregulation of Bax, release of cytochrome c, activation of caspase 3 and 9, and cleavage of PARP. In addition, FZQJ increased the percentages of CD4+ T and NK cells, the ratio of CD4+/CD8+ T cells as well as the levels of serum TNF-α. FZQJ also increased CD69 expression in tumor tissue. No hepatorenal toxicity was observed in H22 tumor-bearing mice. These results indicated that FZQJ could inhibit the growth of hepatoma cells via regulating immune function and inducing mitochondria mediated apoptosis.

Synergisic effect of APRIL knockdown and Jiedu Xiaozheng Yin, a Chinese medicinal recipe, on the inhibition of hepatocellular carcinoma cell proliferation.

It is well documented that A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily, plays a crucial role in the occurrence and development of tumors. In the present study, we evaluated the synergistic effect of APRIL knockdown and Jiedu Xiaozheng Yin (JXY), a Traditional Chinese Medicinal recipe, on the inhibition of hepatocellular carcinoma (HCC) cell proliferation and elucidated the underlying mechanism. The results demonstrated that both APRIL knockdown using small interfering RNA (siRNA) and JXY treatment could trigger cell cycle arrest and cell apoptosis, and suppress HCC cell proliferation through an NF-κB-related pathway. Synergism was further demonstrated between APRIL knockdown and JXY treatment. In conclusion, these results indicate that APRIL is a target gene for HCC and combination of siRNA-APRIL and JXY application holds great promise as a novel approach for the treatment of APRIL-positive HCC.

Ethyl acetate extract of Hypericum japonicum induces apoptosis via the mitochondria-dependent pathway in vivo and in vitro.

The widely-used Chinese medicinal herb Hypericum japonicum, also known as Hypericum japonicum Thunb or Tianjihuang, displays potent anti­carcinogenic effects against liver cancer. However, the molecular mechanism underlying the therapeutic effects of Hypericum japonicum remains to be elucidated. The present study investigated the in vivo efficacy of ethyl acetate extract of Hypericum japonicum (EAEHJ) against tumor growth in an H22 cell­bearing liver cancer mouse model. Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight. The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo. In addition, the anti­carcinogenic effects of EAEHJ were investigated in vitro. The results of the present study demonstrate that both phospholipid asymmetry in the plasma membrane and mitochondrial membrane potential were deregulated in HepG2 human hepatoma cells, following treatment with EAEHJ. Treatment with EAEHJ also increased the ratio of pro­apoptotic B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax) to anti­apoptotic Bcl­2, and activated the caspase­9 signaling pathway. These results suggest that EAEHJ is able to trigger the apoptosis of liver cancer cells via the mitochondria-dependent pathway.

Jiedu Xiaozheng Yin decoction inhibits hepatoma cell proliferation by inducing apoptosis via the mitochondrial-mediated pathway.

Jiedu Xiaozheng Yin decoction (JXY) is a type of Chinese traditional medicine, which has been used to treat various types of cancer. The present study explored the mechanisms underlying the anticancer activity of JXY. The effects of ethyl acetate extraction of JXY (EE-JXY) were evaluated on the HepG2 human hepatoma cell line in vitro and in vivo. Following treatment of the HepG2 cells with EE-JXY for 24 h, cell viability, apoptosis, mitochondrial membrane potential, caspase enzyme activity and the expression levels of apoptotic-associated proteins (Bcl-2 and Bax) were detected by MTT, flow cytometry, ELISA and western blotting respectively. In addition, HepG2 cells were subcutaneously transplanted into BALB/c nude mice, and the tumor bearing mice were treated with either EE-JXY (0.06 g/kg) or normal saline for 21 days. Tumor volume and weight were measured and recorded. The apoptotic index, and the expression levels of Bax and cytochrome c were determined with immunohistochemical staining. Treatment with EE-JXY inhibited the proliferation of HepG2 cells, and reduced cell viability in a dose-and time-dependent manner. Furthermore, EE-JXY induced HepG2 cell apoptosis, as demonstrated by a loss of plasma membrane asymmetry and externalization of phosphatidylserine, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Furthermore, EE-JXY inhibited tumor growth and increased the apoptotic index of tumors in tumor-bearing mice. In conclusion, the results of the present study suggest that JXY inhibits HepG2 cell proliferation through mitochondrion-mediated apoptosis, which may partially explain its anticancer activity.

Active hexose correlated compound potentiates the antitumor effects of low-dose 5-fluorouracil through modulation of immune function in hepatoma 22 tumor-bearing mice.

BACKGROUND/OBJECTIVES: A variety of immunomodulators can improve the efficacy of low-dose chemotherapeutics. Active hexose correlated compound (AHCC), a mushroom mycelia extract, has been shown to be a strong immunomodulator. Whether AHCC could enhance the antitumor effect of low-dose 5-fluorouracil (5-FU) via regulation of host immunity is unknown. MATERIALS/METHODS: In the current study Hepatoma 22 (H22) tumor-bearing mice were treated with PBS, 5-FU (10 mg·kg(-1)·d(-1), i.p), or AHCC (360 mg·kg(-1)·d(-1), i.g) plus 5-FU, respectively, for 5 d. CD3(+), CD4(+), CD8(+), and NK in peripheral blood were detected by flow cytometry. ALT, AST, BUN, and Cr levels were measured by biochemical assay. IL-2 and TNFα in serum were measured using the RIA kit and apoptosis of tumor was detected by TUNEL staining. Bax, Bcl-2, and TS protein levels were measured by immunohistochemical staining and mRNA level was evaluated by RT-PCR. RESULTS: Diet consumption and body weight showed that AHCC had no apparent toxicity. AHCC could reverse liver injury and myelosuppression induced by 5-FU (P < 0.05). Compared to mice treated with 5-FU, mice treated with AHCC plus 5-FU had higher thymus index, percentages of CD3(+), CD4(+), and NK cells (P < 0.01), and ratio of CD4(+)/CD8(+) (P < 0.01) in peripheral blood. Radioimmunoassay showed that mice treated with AHCC plus 5-FU had the highest serum levels of IL-2 and TNFα compared with the vehicle group and 5-FU group. More importantly, the combination of AHCC and 5-FU produced a more potent antitumor effect (P < 0.05) and caused more severe apoptosis in tumor tissue (P < 0.05) compared with the 5-FU group. In addition, the combination of AHCC and 5-FU further up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.01), while it down-regulated the expression of B cell lymphoma 2 (Bcl-2) (P < 0.01). CONCLUSIONS: These results support the claim that AHCC might be beneficial for cancer patients receiving chemotherapy.