Effects of rotation and Bacillus on the changes of continuous cropping soil fungal communities in American ginseng.

The continuous cropping obstacle is the main factor in leading to difficulty in American ginseng replanting. The dormant microbiota in the soil may be the cause of American ginseng disease and eventually caused continuous cropping obstacles, but there are few studies on the dynamic changes of soil microenvironment after American ginseng planting. In this study, we tracked short-term variation in physicochemical properties, enzyme activities, and fungal communities over time-series in soils with continuous cropping obstacle under crop rotation and probiotic Bacillus treatments. Furthermore, we examined the relationships between the important fungal compositions and the soil properties. The results showed that sucrase, cellulase, urease and acid phosphatase activities were significantly increased, while catalase and dehydrogenase were decreased with treatments time. Rotation treatment significantly affected the diversity, dissimilarity degree and species distribution of soil fungal community with continuous cropping obstacle over a short-term. Moreover, beneficial fungal biomarkers such as Cladorrhinum, Oidiodendron, and Mariannaea were accumulated at 48 h under rotation treatments. Almost all fungal biomarkers were negatively correlated with hydrolases and positively correlated with oxidoreductases and acid phosphatase under crop rotation treatments. This study suggested that compared to probiotic Bacillus, crop rotation can significantly affect soil fungal community structure, especially the enrichment of specific potentially beneficial fungal species. Our findings provide a scientific basis for understanding the dynamic changes of fungal communities and soil properties with continuous cropping obstacle of American ginseng in initial stage of soil improvement.

Oxaliplatin resistance is enhanced by saracatinib via upregulation Wnt-ABCG1 signaling in hepatocellular carcinoma.

BACKGROUND: Chemo-resistance in hepatocellular carcinoma (HCC) is a major problem, and acquired drug resistance prevents cancer therapies from achieving complete responses. Molecular targeting therapy presents an opportunity to impede tumor through combination or sequential therapy, while the accurate effect is vague. METHODS: The efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs was screened. Strangely, the combined chemotherapy with oxaliplatin and saracatinib induced significantly antagonistic effects. Then the antitumor effects of combined treatment with saracatinib and oxaliplatin were confirmed in wide type HCC as well as in saracatinib- and oxaliplatin-resistant HCC. RNA sequencing was used to explore the resistance mechanism, and the roles of ATP-binding cassette transporter G1 (ABCG1) and Wnt signaling in oxaliplatin resistance were confirmed. RESULTS: Chemotherapy with oxaliplatin and saracatinib individually induced strong anti-HCC effects, while combined or sequential treatment of HCC cells with these two drugs exhibited reduced efficacy compared to treatment with the single drugs. And it was saracatinib treatment caused oxaliplatin resistance. RNA sequencing revealed 458 genes that were altered by treatment with saracatinib and oxaliplatin. Of these, the gene encoding ABCG1 and Wnt-associated genes were significantly upregulated. Upregulation of ABCG1 and oxaliplatin resistance were associated with activation of Wnt signaling. Interference with ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC. CONCLUSIONS: These studies demonstrated that combined or sequential chemotherapy with oxaliplatin and saracatinib reduced antitumor efficacy, and this antagonism was attributed to the activation of Wnt signaling and upregulation of ABCG1 by saracatinib.

Remodeling of hepatic stellate cells orchestrated the stroma-derived oxaliplatin-resistance through CCN3 paracrine in hepatocellular carcinoma.

BACKGROUND: Hepatic stellate cells (HSCs) have a key role in fibrogenesis and in the filtrates of the hepatocellular carcinoma (HCC) stroma, in which they are remodeled and play a critical role in HCC progression. However, the precise role of HSCs trending, infiltration and paracrine in orchestrating the stroma-derived oxaliplatin-resistance in HCC is still vague. METHODS: The chemo-resistant models were established to explore the correlation between HSC cells and the condition of chemoresistance. The HCC clinical samples were collected to confirm this phenomenon. Then, the relationship between secretory CCN3 from oxaliplatin-resistant HCC and the infiltration of HSCs in associated HCC microenvironment was evaluated. Finally, the role and mechanism of HSCs remodeling in the orchestration of oxaliplatin-resistant HCC were explored. RESULTS: The increased infiltration of HSCs and collagen accumulation were found in the microenvironment of oxaliplatin-resistant HCC. The cDNA profiles of the oxaliplatin-resistant HCC was reanalyzed, and CCN3 was one of the significantly increased genes. In HCC clinical samples, the levels of CCN3 and α-SMA are positively correlated, and high expression of CCN3 and α-SMA are positively associated with malignant phenotype and poor prognosis. Then the enhanced abilities of migration and proliferation of HSCs, and elevation of the cytokines paracrine from HSCs relating to HCC malignancy were proved in vitro and in vivo, and which were related to CCN3-ERK signaling pathway activation. CONCLUSIONS: HSCs remodeling are positively related to CCN3 paracrine in hepatocellular carcinoma, which orchestrated the stroma-derived resistance to chemotherapy in HCC.

Paracrine effects of CCN3 from non-cancerous hepatic cells increase signaling and progression of hepatocellular carcinoma.

BACKGROUND: The liver microenvironment plays a key role in the progression and metastasis of hepatocellular carcinoma (HCC). Gene expression profiling of non-cancerous hepatic tissues obtained from patients with metastatic HCC exhibit a unique immune response signature, including upregulation of CCN3. However, the role of CCN3 secreted from non-cancerous hepatic tissues in the progression of HCC remains unclear. METHODS: Using tissue microarrays, we examined CCN3 in non-cancerous hepatic tissues of patients with HCC and correlated expression with clinical and pathological features. In addition, CCN3 localization and mechanisms of HCC progression were investigated in tissues and cell lines. Finally, correlations between CCN3 and cirrhosis were explored in patients. RESULTS: CCN3 was primarily localized to hepatic cells of non-cancerous hepatic tissues and was associated with vascular invasion and poor prognosis in patients with HCC. CCN3 expression in non-cancerous hepatic tissues also correlated with the degree of liver fibrosis. Compared with conditioned media from wild-type LO2 cells, conditioned media from hepatic cell line LO2 activated by LX2 (aLO2-CM) induced CCN3 expression and HCC cell proliferation and metastasis. Further, aLO2-CM activated MAPK signaling and epithelial-mesenchymal transition in HCC cells. Finally, CCN3 was inversely related to cirrhosis in the prognosis of HCC and negatively regulated hepatic stellate cells (HSCs) in vitro with downregulation of α-SMA, TGF-ß, and collagens. CONCLUSIONS: CCN3 was secreted from hepatic cells activated by HSCs and increased MAPK signaling, EMT, proliferation and metastasis of HCC cells. CCN3 was also inversely related to cirrhosis, regulating HSCs through a negative feedback loop.

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis.

BACKGROUND/AIMS: Effective drug treatment for intrahepatic cholangiocarcinoma (ICC) is currently lacking. Therefore, there is an urgent need for new targets and new drugs that can prolong patient survival. Recently targeting the ubiquitin proteasome pathway has become an attractive anti-cancer strategy. In this study, we aimed to evaluate the therapeutic effect of and identify the potential mechanisms involved in targeting the proteasome subunit ADRM1 for ICC. METHODS: The expression of ADRM1 and its prognostic value in ICC was analyzed using GEO and TCGA datasets, tumor tissues, and tumor tissue arrays. The effects of RA190 on the proliferation and survival of both established ICC cell lines and primary ICC cells were examined in vitro. Annexin V/propidium iodide staining, western blotting and immunohistochemical staining were performed. The in vivo anti-tumor effect of RA190 on ICC was validated in subcutaneous xenograft and patient-derived xenograft (PDX) models. RESULTS: ADRM1 levels were significantly higher in ICC tissues than in normal bile duct tissues. ICC patients with high ADRM1 levels had worse overall survival (hazard ratio [HR] = 2.383, 95% confidence interval [CI] =1.357 to 4.188) and recurrence-free survival (HR = 1.710, 95% CI =1.045 to 2.796). ADRM1 knockdown significantly inhibited ICC growth in vitro and in vivo. The specific inhibitor RA190 targeting ADRM1 suppressed proliferation and reduced cell vitality of ICC cell lines and primary ICC cells significantly in vitro. Furthermore, RA190 significantly inhibited the proteasome by inactivating ADRM1, and the consequent accumulation of ADRM1 substrates decreased the activating levels of NF-κB to aggravate cell apoptosis. The therapeutic benefits of RA190 treatment were further demonstrated in both subcutaneous implantation and PDX models. CONCLUSIONS: Our findings indicate that up-regulated ADRM1 was involved in ICC progression and suggest the potential clinical application of ADRM1 inhibitors (e.g., RA190 and KDT-11) for ICC treatment.

Targeting mTORC1/2 Complexes Inhibit Tumorigenesis and Enhance Sensitivity to 5-Flourouracil (5-FU) in Hepatocellular Carcinoma: A Preclinical Study of mTORC1/2-Targeted Therapy in Hepatocellular Carcinoma (HCC).

BACKGROUND Although 5-Flourouracil(5-FU) is used as the first-choice treatment for advanced hepatocellular carcinoma (HCC), it is associated with acquired and intrinsic resistance. Hyperactivation of mTOR signaling has been linked to tumorigenesis and chemoresistance in HCC. The aim of this study was to evaluate and compare the antitumor effects of mTORC1 inhibitor everolimus and mTORC1/2 inhibitor AZD8055 and to examine the interaction between 5-FU and mTORC1/2 inhibitor in HCC. MATERIAL AND METHODS Using cultured HCC cells and mouse xenograft, the antitumor effects of everolimus and AZD8055 were analyzed as mono- and combination therapy with 5-Flourouracil. RESULTS TSC2-deficient HCC cell lines were more sensitive to everolimus and AZD8055. AZD8055, but not everolimus, potently prevented cells from transitioning from G1 phase to S phase in TSC2-high-expressing HCC cells. AZD8055 reduced phosphorylation of both mTORC1 and mTORC2 substrates. In contrast, everolimus reduced the phosphorylation of mTORC1 substrates, but increased the phosphorylation of AKT. Notably, AZD8055, but not everolimus, synergistically enhanced the efficacy of 5-FU via reversing 5-FU-induced upregulation of P-glycoprotein (P-gp). The synergistic antitumor effect of AZD8055 and 5-FU was also observed in a HCC xenograft mouse model. CONCLUSIONS TSC2 in HCC is a promising efficacy-predicting biomarker for the treatment of mTORC1/2 inhibitor. AZD8055 showed stronger antitumor activity than everolimus in TSC2-high-expressing HCC cells. Moreover, the combination of mTORC1/2 inhibitor with 5-FU appears to be a promising option for HCC patients refractory to chemotherapy.

S100A6 promotes proliferation of intrahepatic cholangiocarcinoma cells via the activation of the p38/MAPK pathway.

AIM: We explored the expression of S100A6 and its role in intrahepatic cholangiocarcinoma (ICC). METHODS: The expression of S100A6 in ICC samples was detected by immunohistochemistry. In vitro experiments, we silenced and overexpressed S100A6 to investigate its role in cell functions. RESULTS: The expression of S100A6 was markedly increased in ICC tissues and cell lines. S100A6 overexpression was an independent risk factor for patients' survival. Silencing S100A6 resulted in a suppression of proliferation and p38/MAPK activity, while overexpressing S100A6 caused a promotion of proliferation and p38/MAPK. DISCUSSION:  S100A6 participated in the proliferation of ICC cells and correlated with a more aggressive behavior of ICC. Conclusion: S100A6 may serve as a novel prognostic marker and a potential therapeutic target for ICC patients.

High-mobility group protein box1 expression correlates with peritumoral macrophage infiltration and unfavorable prognosis in patients with hepatocellular carcinoma and cirrhosis.

BACKGROUND: High-mobility group protein box1 (HMGB1) is a pivotal factor in the development and progression of many types of tumor. Its role in hepatocellular carcinoma (HCC), and especially its correlation with intratumoral and peritumoral macrophage infiltration, remains obscure. We analyzed the potential roles and prognostic value of HMGB1 and explored the correlation between HMGB1 and macrophage infiltration in HCC using clinical samples. METHODS: We reviewed clinicopathological and follow-up data on a cohort of 149 patients with HCC complicated with Hepatitis B-related cirrhosis. We measured the expression of HMGB1 and CD68 in tumoral and peritumoral liver tissues after curative resection and assessed the impacts of the tumor-associated macrophage (TAM) count and HMGB1 expression on clinicopathologic characteristics, overall survival (OS), and recurrence-free survival (RFS). RESULTS: Ninety-four of the patients had elevated tumoral HMGB1 expression and 59 of the patients had elevated peritumoral HMGB1 expression, compared to only 4 patients with elevated peritumoral HMGB1 expression in 36 pateints with Hepatitis B virus (HBV)-negative HCC without liver cirrhosis (p < 0.001). The peritumoral HMGB1 expression levels were correlated with tumor invasiveness, BCLC stage, and recurrence. The degree of TAM infiltration was higher in peritumoral tissues with high HMGB1 expression than in peritumoral tissues with low HMGB1 expression (p < 0.001). There was no significant difference in TAM infiltration between tumoral tissues with high and low HMGB1 expression. Kaplan-Meier analysis showed that intratumoral HMGB1 overexpression was associated with poor OS, but not with RFS. High peritumoral HMGB1expression and TAM count, which correlated positively with tumor size and BCLC stage, were independent prognostic factors for OS (p < 0.001 and p = 0.017, respectively) and RFS (p = 0.002 and p = 0.024, respectively). Multivariate analyses indicated peritumoral HMGB1 expression (p = 0.014) and TAM count (p = 0.037), as well as tumor differentiation (p = 0.026), to be independent significant prognostic factors for RFS. CONCLUSIONS: High HMGB1 expression in peritumoral liver tissues correlated with peritumoral macrophage infiltration and had prognostic value in HCC, suggesting that peritumoral HMGB1 might show promise as a new biomarker to predict HCC progression.

Imbalance in systemic inflammation and immune response following transarterial chemoembolization potentially increases metastatic risk in huge hepatocellular carcinoma.

Inflammation plays a critical role in tumor metastasis. However, few inflammation-related biomarkers are currently available to predict the risk of metastasis for advanced hepatocellular carcinoma (HCC). Using huge tumors (diameter >10 cm) as a model, we evaluated the potential risk of pre- and post-treatment inflammatory responses in the development of metastasis of HCC patients undergoing transarterial chemoembolization (TACE). A logistic regression model was used to analyze the risk factors. One hundred and sixty-five patients with huge HCC were enrolled in the study. Metastases were identified in 25.5% (42/165) patients by imaging evaluation post-TACE. Neutrophils increased, whereas lymphocytes decreased significantly post-TACE. Univariate analysis showed that high post-treatment neutrophil-to-lymphocyte ratio (NLR; p = 0.003), low post-treatment lymphocyte count (p = 0.047), and high baseline NLR (p = 0.100) were potential risk factors for metastasis. Further, multivariate analysis showed that high post-treatment NLR, but not pre-treatment NLR, was an independent risk factor for metastasis; this was confirmed by receiver operating characteristic curve analysis. Post-treatment NLR, however, had no correlation to tumor response and overall survival of patients. In conclusion, post-treatment NLR but not pre-treatment NLR independently increases the risk of metastasis in huge HCC. Our findings suggest the potential contribution of treatment-related inflammation to metastasis in advanced HCC.

The platelet-to-lymphocyte ratio predicts poor survival in patients with huge hepatocellular carcinoma that received transarterial chemoembolization.

Inflammation is particularly strong in huge hepatocellular carcinoma (HCC). However, it is unclear whether the platelet-to-lymphocyte ratio (PLR), as an inflammatory-related marker, can predict survival of patients with huge HCC. In this study, we enrolled 291 patients with huge HCC (diameter over 10 cm) who were undergoing repeated transarterial chemoembolization (TACE) at our institute. The baseline PLR was calculated from complete serum blood counts before the first chemoembolization. We found that a baseline PLR cutoff value over 150 best predicted huge HCC survival. The 12, 24, and 36 months survival rates in the high PLR group (22.6, 8.1, and 4.1 %, respectively) were significantly lower than in the low PLR group (35.6, 22.4, and 14 %, respectively). Thus, a significant difference was found in overall survival (log-rank test, p < 0.0001). Univariate analyses indicated a high PLR (p < 0.0001) was predictor of poor survival, and multivariate Cox analyses further showed that a high PLR (p = 0.002) was an independent factor that predicted worse survival. In conclusion, for patients with huge HCC, a high baseline PLR is a useful predictor of poor survival in patients undergoing chemoembolization. Additional anti-inflammatory or anti-platelet treatments, in combination with TACE, may improve survival in HCC patients with high PLR.

Inositol hexaphosphate enhances chemotherapy by reversing senescence induced by persistently activated PERK and diphthamide modification of eEF2.

Oxaliplatin is an important initial chemotherapy benefiting advanced-stage colorectal cancer patients. Frustratingly, acquired oxaliplatin resistance always occurs after sequential chemotherapy with diverse antineoplastic drugs. Therefore, an exploration of the mechanism of oxaliplatin resistance formation in-depth is urgently needed. We generated oxaliplatin-resistant colorectal cancer models by four representative compounds, and RNA-seq revealed that oxaliplatin resistance was mainly the result of cells' response to stimulus. Moreover, we proved persistent stimulus-induced endoplasmic reticulum stress (ERs) and associated cellular senescence were the core causes of oxaliplatin resistance. In addition, we screened diverse phytochemicals for ER inhibitors in silico, identifying inositol hexaphosphate (IP6), whose strong binding was confirmed by surface plasmon resonance. Finally, we confirmed the ability of IP6 to reverse colorectal cancer chemoresistance and investigated the mechanism of IP6 in the inhibition of diphthamide modification of eukaryotic elongation factor 2 (eEF2) and PERK activation. Our study demonstrated that oxaliplatin resistance contributed to cell senescence induced by persistently activated PERK and diphthamide modification of eEF2 levels, which were specifically reversed by combination therapy with IP6.

Schedule-dependent antitumor effects of 5-fluorouracil combined with sorafenib in hepatocellular carcinoma.

BACKGROUND: Recently, a phase II clinical trial in hepatocellular carcinoma (HCC) has suggested that the combination of sorafenib and 5-fluorouracil (5-FU) is feasible and side effects are manageable. However, preclinical experimental data explaining the interaction mechanism(s) are lacking. Our objective is to investigate the anticancer efficacy and mechanism of combined sorafenib and 5-FU therapy in vitro in HCC cell lines MHCC97H and SMMC-7721. METHODS: Drug effects on cell proliferation were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle. Cell cycle distribution was measured by flow cytometry. Expression levels of proteins related to the RAF/MEK/ERK and STAT3 pathways and to cell cycle progression (cyclin D1) were determined by western blot analysis. RESULTS: Sorafenib and 5-FU alone or in combination showed significant efficacy in inhibiting cell proliferation in both cell lines tested. However, a schedule-dependent combined effect, associated with the order of compound treatments, was observed. Efficacy was synergistic with 5-FU pretreatment followed by sorafenib, but it was antagonistic with the reverse treatment order. Sorafenib pretreatment resulted in a significant increase in the half inhibitory concentration (IC50) of 5-FU in both cell lines. Sorafenib induced G1-phase arrest and significantly decreased the proportion of cells in S phase when administrated alone or followed by 5-FU. The RAF/MEK/ERK and STAT3 pathways were blocked and cyclin D1 expression was down regulated significantly in both cell lines by sorafenib; whereas, the kinase pathways were hardly affected by 5-FU, and cyclin D1 expression was up regulated. CONCLUSIONS: Antitumor activity of sorafenib and 5-FU, alone or in combination, is seen in HCC cell lines. The nature of the combined effects, however, depends on the particular cell line and treatment order of the two compounds. Sorafenib appears to reduce sensitivity to 5-FU through down regulation of cyclin D1 expression by inhibiting RAF/MEK/ERK and STAT3 signaling, resulting in G1-phase arrest and reduction of the S-phase cell subpopulation when 5-FU is administrated after sorafenib, in which situation, combination treatment of the two agents results in antagonism; on the other hand, when sorafenib is administrated afterward, it can continue to work since it is not cell cycle specific, as a result, combination treatment of the two agents shows an additive-to-synergistic effect.

Herbal compound "Songyou Yin" attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells.

BACKGROUND: Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound "Songyou Yin" (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells. METHODS: Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-ß1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY. CONCLUSIONS: SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells.

Effect of the GLP-1 analog exendin-4 and oxaliplatin on intrahepatic cholangiocarcinoma cell line and mouse model.

The influence of Glucagon-like peptide-1 (GLP-1) and Exendin-4 on development of intrahepatic cholangiocarcinoma (ICC) is evaluated in the study. In vitro tests, including acute toxicity test, cell colony formation assays, cells proliferation and apoptosis, transwell assay, were performed. An ICC in situ tumor animal model was established. Then, animals were randomly divided into four groups (n = 6): control, Exendin-4 treatment, oxaliplatin treatment and Exendin-4-oxaliplatin treatment. Animals in the Exendin-4 treatment and Exendin-4-oxaliplatin treatment groups received a subcutaneous injection of Exendin-4 (100 µg/kg/day) for 1 week, and then received oxaliplatin (10 mg/kg/week) by tail vein injection. Animals in the control group received PBS. Immunohistochemistry tests were used for PCNA, Ki67, Caspase 3 expression in tumor tissue. Results show that that, after incubation of human cholangiocarcinoma cell lines, HuCCTI and GLP-1, or HuCCTI and Exendin-4, colony formation number was sharply decreased. However, GLP-1, HuCCTI or Exendin-4 did not affect the colony of normal cells. Combination treatment with oxaliplatin and Exendin-4 can significantly inhibit tumor cells' proliferation and promote apoptosis. The combined effect is stronger than that of oxaliplatin or Exendin-4. Combination treatment with oxaliplatin and Exendin4 can significantly decrease Ki67 and PCNA proteins' expression in subcutaneous tumors of nude mice. The inhibitory effect of Combination treatment with oxaliplatin and Exendin4 is clearly stronger than that of oxaliplatin. In addition, Combination treatment with oxaliplatin and Exendin4 can significantly increase Caspase3 protein positive expression. In short, these results show that combination treatment with oxaliplatin and Exendin4 can inhibit tumor cells' proliferation, and promote apoptosis.

[Effects of the Chinese herbal extract Songyou Yin on the residual hepatocellular carcinoma after chemotherapy in nude mice].

OBJECTIVE: To investigate the effects of a Chinese herbal extract Songyou Yin on residual hepatocellular carcinoma after chemotherapy in nude mice and the relevant mechanisms. METHODS: Orthotopic nude mouse models bearing residual hepatocellular carcinoma after chemotherapy was established using human liver carcinoma MHCC97L cells. Three different doses of Songyon Yin (2.1 g/kg, 4.2 g/kg and 8.4 g/kg) were administered to the mice in the trial groups by intragastric gavage, respectively. The mice in the control group were administered physiological saline. The tumor growth, metastasis and survival in the mice of each group were recorded. The corresponding mechanisms were studied. RESULTS: The pulmonary metastasis rates of the control group and 2.1g/kg, 4.2g/kg, 8.4g/kg Songyou Yin treatment group were 86.7%, 73.3%, 40.0%, and 20.0%, respectively, and the survivals of these groups were 53.83 ± 4.71, 56.50 ± 6.09, 66.67 ± 5.61, 81.17 ± 7.36 days, respectively. Compared with the mice in the control group, mice in the 4.2 g/kg, 8.4 g/kg Songyou Yin treatment groups had a lower pulmonary metastasis rate (P = 0.021 and P = 0.001, respectively) and longer survival (P = 0.002 and P = 0.001, respectively). A restoration of E-cadherin expression and a concomitant reduction of N-cadherin expression were detected in the tumors of the 4.2 g/kg and 8.4 g/kg Songyou Yin treatment groups. CONCLUSIONS: Songyou Yin effectively inhibits the invasion and metastasis of the residual hepatocellular carcinoma after chemotherapy in nude mice through attenuating the epithelia-mesenchymal transition and prolongs the survival. Songyon Yin may have potential to promote the efficacy of chemotherapy in hepatocellular carcinoma.

[Anti-proliferation effect of sorafenib in combination with 5-FU for hepatocellular carcinoma in vitro: antagonistic performance and mechanism].

OBJECTIVE: To investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3. METHODS: The effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting. RESULTS: Sorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates: sorafenib: 46.16% +/- 2.52%, 5-FU: 28.67% +/- 6.16%, and sorafenib + 5-FU: 22.59% +/- 6.89%. The sorafenib + 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 +/- 27.84) mg/L to (178.61 +/- 20.73) mg/L (P = 0.003). Sorafenib alone induced G1 phase arrest (increasing from 44.73% +/- 1.63% to 65.80% +/- 0.56%; P less than 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63% +/- 0.65% to 22.83% +/- 1.75%; P less than 0.01), as well as down-regulated cyclinD1 expression (0.57 +/- 0.03-fold change vs. untreated control group; P less than 0.01). 5-FU alone up-regulated cyclinD1 expression (1.45 +/- 0.12-fold change vs. untreated control group; P less than 0.01). Moreover, sorafenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways, with the fold-changes of p-C-RAF, p-ERK1/2 and p-STAT3 being 0.56 +/- 0.05, 0.54 +/- 0.02 and 0.36 +/- 0.02, respectively (all P less than 0.01); 5-FU alone produced no significant effects on these pathways. CONCLUSION: Administered alone, both sorafenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells. The sorafenib + 5-FU combination treatment produces antagonistic, rather than synergistic, effects. Sorafenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G1phase arrest and S phase reduction, thereby reducing the cells' sensitivity to 5-FU.

MiR-128-1-5p inhibits cell proliferation and induces cell apoptosis via targeting PRKCQ in colorectal cancer.

Previous studies have indicated that miR-128 was downregulated in a variety of cancers including colorectal cancer (CRC). However, the role and the underlying molecular mechanisms of miR-128 in CRC still remain largely unknown. The aim of this study was to investigate the level of miR-128-1-5p in CRC patients and to explore both the effects and regulatory mechanisms of miR-128-1-5p in the malignancy of CRC. Real-time PCR and western blot were used to analyze the expression levels of miR-128-1-5p and the direct downstream target protein tyrosine kinase C theta isoform (PRKCQ). Cell Counting Kit-8, clone formation, TUNEL apoptosis assays, and subcutaneous tumor model were performed to investigate the malignant ability of colon cancer cells. A luciferase assay was performed to explore whether miR-128-1-5p could directly bind to 3'-UTR region of PRKCQ. In the present study, we detected the decreased expression and clinical significances of miR-128-1-5p in colorectal cancer tissues and cell lines. Functional experiments revealed that miR-128-1-5p inhibited cell proliferation and induced cell apoptosis and that PRKCQ was identified as a target of miR-128-1-5p and involved in miR-128-1-5p-mediated proliferation and apoptosis. In conclusion, our results showed that miR-128-1-5p reduced CRC growth by modulating PRKCQ expression and is a possible new therapeutic target for patients with CRC.

Herbal Compound "Songyou Yin" Renders Hepatocellular Carcinoma Sensitive to Oxaliplatin through Inhibition of Stemness.

We investigated the effect of Chinese herbal compound Song-you Yin on HCC stemness. MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks, and their chemosensitivity to oxaliplatin was evaluated. The expression of CSC-related markers, cell invasion and migration, and colony formation were also examined. SYY-treated orthotopic nude mouse models of human HCC were developed to explore the effect of oxaliplatin on tumor growth, metastasis, and survival. The CSC-related molecular changes in vivo were also evaluated. The result showed that MHCC97H and Hep3B cells pretreated with SYY showed significantly increased chemosensitivity to oxaliplatin and the downregulation of CSC-related markers CD90, CD24, and EPCAM. SYY also attenuated cell motility, invasion, and colony formation in MHCC97H and Hep3B cell lines. The reduced tumorigenicity and pulmonary metastasis were observed in SYY-pretreated cell lines. Combination treatment with oxaliplatin and SYY significantly reduced tumor volume and pulmonary metastasis and prolonged survival compared with oxaliplatin treatment alone. Immunohistochemical analysis showed reduced expression of CD90, ABCG2, ALDH, CD44, EPCAM, vimentin, and MMP-9 and increased the expression of E-cadherin, in HCC cells following combination treatment. These data clearly demonstrate that SYY renders hepatocellular carcinoma sensitive to oxaliplatin through the inhibition of stemness.

Anti-Tumor Role of CAMK2B in Remodeling the Stromal Microenvironment and Inhibiting Proliferation in Papillary Renal Cell Carcinoma.

The tumor microenvironment (TME) is variable across tumor types and has diverse effects on malignant progression, based on the type and number of infiltrating stromal cells. In particular, TME effector genes and their competitive endogenous RNA (ceRNA) networks play a critical role in regulating malignant tumor progression. However, the core effector molecules involved in TME modulation of kidney renal papillary cell carcinoma (KIRP) are poorly understood. To address this question, a cohort containing 233 KIRP patients was derived from The Cancer Genome Atlas (TCGA) database, and the data were processed using the ESTIMATE algorithm. We further evaluated the relationship between immune scores (ISs) and stromal scores (SSs) and disease progression and found that high SSs were associated with a poor prognosis in KIRP. Differentially expressed genes (DEGs) were therefore screened based on SS scores, resulting in 2509 DEGs, including 1668 mRNAs, 783 long noncoding (lnc)RNAs, and 58 micro (mi)RNAs. DEGs were then filtered using the random variance and subjected to hierarchical clustering using EPCLUST. Weighted gene co-expression network analysis (WGCNA) was used to assess the prognostic capacity of these DEGs and identify target ceRNA networks, and lncRNA GUSBP11/miR-432-5p/CAMK2B in the turquoise module was selected as a promising ceRNA network. From this analysis CAMK2B was selected as the core gene predicted to be involved in stromal TMA regulation. We therefore explored the expression and function of CAMK2B in vitro and in vivo and provide evidence that this protein promotes stromal TME remodulation and inhibits proliferation in KIRP. Lastly, we show that vascular endothelial growth factor (VEGF), transforming growth factor (TGF)ß, and close homolog of L1 (CHL1) act as downstream effectors of CAMK2B in KIRP. Thus, in this study, we show that the TME determines prognosis of KIRP patients via the core effector molecule CAMK2B, which mediates both microenvironmental remodeling and tumor progression. Based on these findings, we propose that remodeling of the stromal microenvironment could represent an improved therapeutic approach relative to immunotherapy for KIRP.

Responses of Soil Rare and Abundant Sub-Communities and Physicochemical Properties after Application of Different Chinese Herb Residue Soil Amendments.

Microbial diversity in the soil is responsive to changes in soil composition. However, the impact of soil amendments on the diversity and structure of rare and abundant sub-communities in agricultural systems is poorly understood. We investigated the effects of different Chinese herb residue (CHR) soil amendments and cropping systems on bacterial rare and abundant sub-communities. Our results showed that the bacterial diversity and structure of these sub-communities in soil had a specific distribution under the application of different soil amendments. The CHR soil amendments with high nitrogen and organic matter additives significantly increased the relative abundance and stability of rare taxa, which increased the structural and functional redundancy of soil bacterial communities. Rare and abundant sub-communities also showed different preferences in terms of bacterial community composition, as the former was enriched with Bacteroidetes while the latter had more Alphaproteobacteria and Betaproteobacteria. All applications of soil amendments significantly improved soil quality of newly created farmlands in whole maize cropping system. Rare sub-communitiy genera Niastella and Ohtaekwangia were enriched during the maize cropping process, and Nitrososphaera was enriched under the application of simple amendment group soil. Thus, Chinese medicine residue soil amendments with appropriate additives could affect soil rare and abundant sub-communities and enhance physicochemical properties. These findings suggest that applying soil composite amendments based on CHR in the field could improve soil microbial diversity, microbial redundancy, and soil fertility for sustainable agriculture on the Loess Plateau.