Histomorphological and molecular genetic characterization of different intratumoral regions and matched metastatic lymph nodes of colorectal cancer with heterogenous mismatch repair protein expression.

PURPOSE: To better understand the clinicopathological characteristics and molecular alterations in different intratumoral components of colorectal cancer (CRC) with heterogeneity of mismatch repair (MMR) protein expression and microsatellite instability (MSI) status. METHODS: The histopathological features, MSI status, and other molecular alterations were analyzed in separately microdissected intratumoral regions and matched metastatic lymph nodes in four cases with intratumoral heterogenous MMR expression screened from 500 CRC patients, using PCR-based MSI testing, MLH1 promoter methylation, and targeted next-generation sequencing (NGS). RESULTS: High microsatellite instability (MSI-H) was identified in MLH1/PMS2-deficient regions in Cases 1 to 3 and in MSH2/MSH6-deficient regions in Case 4, while microsatellite stability (MSS) was detected in all the intratumoral regions and metastatic lymph nodes with proficient MMR expression (pMMR). Intratumoral heterogeneity of MLH1 promoter methylation and/or other common driving gene mutations of CRC, such as KRAS and PIK3CA mutations, was identified in all four CRCs. Further, three cases (75%) showed heterogeneous histomorphological features in intratumoral components and metastatic lymph nodes (Cases 1, 2, and 4), and the corresponding metastatic lymph nodes showed moderate differentiation with MSS/pMMR (Cases 2 and 3). CONCLUSIONS: Intratumoral heterogeneous MSI status is highly correlated with intratumoral histomorphological heterogeneity, which is also an important clue for the intratumoral heterogeneity of drive gene mutations in CRC. Thus, it is essential to detect MMR protein expression and other gene mutations in metastases before treatment, especially for CRCs with intratumoral heterogenous MMR protein expression or heterogenous histomorphological features.

Role of the prostaglandin E2 receptor agonists in TGF-ß1-induced mesangial cell damage.

PGE2 exerts its biological effect through binding to various EP receptors that result inactivation of various signal transduction pathways. It also plays an important role in mice glomerular mesangial cells (MCs) damage induced by transforming growth factor-ß1 (TGF-ß1); however, the molecular mechanisms remain unknown. In the present study, we tested the efficacy of four selective agonists of PGE2 receptor, EP1A (17-phenyl trinor prostaglandin E2 ethyl amid), EP2A (butaprost), EP3A (sulprostone) and EP4A (cay10580), on mice MCs. Compared with the cAMP produced by TGF-ß1, additional pretreatment of EP3A decreased the cAMP level. MCs treated with EP1A and EP3A augmented PGE2, cyclooxygenase-2 (COX-2), membrane-bound PGE synthase 1 (mPGES1), laminin (LN), connective tissue growth factor (CTGF) and cyclin D1 expression stimulated by TGFß1. EP1A and EP3A increased the number of cells in S+G2/M phase and reduced cells in G0/G1 phase. EP1 and EP3 agonists also strengthened TGFß1-induced mitogen-activated protein kinase (p38MAPK) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Whereas MCs treated with EP2A and EP4A weakened PGE2, COX-2, mPGES1, LN, CTGF and cyclin D1 expression stimulated by TGFß1. EP2A and EP4A decreased the number of cells in S+G2/M phase and increased cells in G0/G1 phase. EP2 and EP4 agonists weakened TGFß1-induced p38MAPK and ERK1/2 phosphorylation. These findings suggest that PGE2 has an important role in the progression of kidney disease via the EP1/EP3 receptor, whereas EP2 and EP4 receptors are equally important in preserving the progression of chronic kidney failure. Thus, agonists of EP2 and EP4 receptors may provide a basis for treating kidney damage induced by TGF-ß1.

TLR3 Plays Significant Roles against HBV-Associated HCC.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). The role of TLR3 in hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) infections is not well understood. To investigate the ability of TLR3 in regulating HBV replication in HCC, 80 cases of human HCC were collected and their tissue microarray was made. In HCC cells, the expression and location of TLR3, hepatitis-associated virus, and interstitial immunoreactive cells were assayed with immunohistochemical staining. The apoptosis of tumor cells was also detected by TUNEL stain. Correlations between TLR3 expression and HBV infection, interstitial immunoreactive cells, and cells apoptosis in HCC were investigated. In addition, we explored whether TLR3 agonist dsRNA can inhibit HepG2.2.15 cells secreting HBV. We found that the cytoplasmic expression of TLR3 in HCC is positively related to HBsAg infection and HCC with cirrhosis and promotes interstitial immunoreactive cells infiltration and cancer cells apoptosis. In HepG2.2.15 cells, dsRNA inhibited the secretion of HBV and induced apoptosis. These results indicate that TLR3 signaling activity may be involved in immune responses against HBV in HCC.

TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis.

BACKGROUND: Toll-like receptor 3 (TLR3) plays a key role in innate immunity. In the present study, we analyzed tissues of patients with human hepatocellular carcinoma (HCC) to determine the significance of the relationship between TLR3 expression and cell proliferation, apoptosis, hepatitis B virus infections, angiogenesis and prognosis. METHODS: We collected paraffin-embedded tissues from 85 patients with HCC who had complete histories and were followed for >5 years. The expression and intracellular localization of TLR3 and downstream proteins (TRIF, NF-κB, and IRF3) were detected using immunohistochemistry. Further, we determined the expression of proteins that mediate cell proliferation (Ki67, cyclin D1), apoptosis (survivin, bcl-2, caspases 3, 8, and 9), and angiogenesis (CD34, MMP-2) as well as the HBV proteins HBsAg and HBcAg. Apoptosis in HCC tissues was detected using TUNEL. We conducted dual-labeling immunohistochemical analyses of TLR3 expression and TUNEL activity. RESULTS: TLR3 expression was significantly lower in HCC tissues compared with adjacent tissues. TRIF, NF-κB, and IRF3 correlated positively with TLR3 expression. Survivin and Bcl-2 expression correlated negatively with TLR3. The frequencies of caspases 3, 8, and 9 expression correlated positively with TLR3 signaling proteins. Cytoplasmic TLR3 and serum levels of HBsAg correlated positively. The apoptotic index determined using the TUNEL method and correlated positively with TLR3 expression. TLR3 expression in the cytoplasm correlated positively with TUNEL-positive cells and HBsAg. Ki67 and cyclin D1 correlated negatively with TLR3 expression. MMP-2 expression, microvessel density (CD34(+)) and endothelial progenitor cells (EPCs) correlated negatively with TLR3 expression. Kaplan-Meier survival analysis shows that TLR3 expression correlated with longer survival. CONCLUSIONS: The expression of TLR3 in HCC tissues may exert a synergistic effect on apoptosis and inhibit the proliferation of HCC cells, MMP-2 expression, generation of EPCs, and angiogenesis. Moreover, TLR3 expression may serve as a prognostic marker of HCC.

Role of the prostaglandin E2/E-prostanoid 2 receptor signalling pathway in TGFß-induced mice mesangial cell damage.

The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFß1 (transforming growth factor-ß1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFß1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E2 synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFß1 were increased; however, the production of cAMP and PGE2 (prostaglandin E2) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFß1. Silencing of EP2 also strengthened TGFß1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE2 through EP2 receptors which prevent TGFß1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFß1.

A maladaptive role for EP4 receptors in mouse mesangial cells.

Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4) on extracellular matrix (ECM) accumulation induced by TGF-ß1 in mouse glomerular mesangial cells (GMCs) remain unknown. Previously, we have identified that TGF-ß1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4(Flox/Flox) and EP4(+/-) mice, cultured primary WT, EP4(Flox/Flox) and EP4(+/-) GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression) and AD-Cre transfected EP4(Flox/Flox) GMCs (EP4 deleted). We found that TGF-ß1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-ß1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-ß1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx), WT and EP4(+/-) mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr) concentrations were significantly increased in WT mice as compared to those of EP4(+/-) mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4(+/-) mice. The pathological changes in kidney of EP4(+/-) mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4(+/-) mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-ß1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.

The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance.

BACKGROUND: A role for autophagy, a conserved cellular response to stress, has recently been demonstrated in human cancers. Aberrant expression of Beclin-1, an important autophagic gene, has been reported in various human cancers. In the present study, we investigated the significance and relationship between Beclin-1 expression and cell proliferation, apoptosis, microvessel density (MVD) and clinical pathological changes or prognosis in human hepatocellular carcinoma (HCC). METHODS: A total of 103 primary HCC patients were involved in the study. Expression of Beclin-1, PCNA, NET-1, Bcl-2, Bax, Survivin in cancer cells and CD34 in stromal microvessels were evaluated immunohistochemically in tissue microarrays comprising 103 cases of HCC and 57 matched adjacent nontumor liver tissues. Correlations between clinicopathological characteristics and survival of HCC patients were explored. RESULTS: The positive rate of Beclin-1 was significantly lower in HCC tissues than adjacent tissues (72.8 vs. 89.5%, χ2 = 6.085, P = 0.015). In HCC, Beclin-1 expression was negatively correlated with cirrhosis background (r = -0.216, P = 0.029), Edmondson grade (r = -0.249, P = 0.011), vascular invasion (r = -0.246, P = 0.012), PCNA (r = -0.242, P = 0.014), NET-1 (r = -0.245, P = 0.013), anti-apoptosis protein Bcl-2 (r = -0.245, P = 0.013) and MVD (r = -0.292, P = 0.003), and positively correlated with pro-apoptosis protein Bax (r = 0.242, P = 0.014).Significant differences in the 5-year survival rates were seen among patients with Beclin-1 strong positive (++) (59.1%, 13/22), moderate positive (+) (28.3%, 15/53) and weak negative expression (-) (14.6%, 7/28) (P = 0.043). Significant differences were detected between Beclin-1 (++) and either Beclin-1 (+) (P = 0.036) or Beclin-1 (-) groups (P = 0.008), but no significant difference between Beclin-1 (+) and Beclin-1 (-) groups (P = 0.281) was observed.Survival rates were positively related to high Beclin-1 co-expressed with low PCNA, NET-1, or Bcl-2, lower MVD, and high Bax. Univariate and multivariate Cox regression analysis revealed that Beclin-1 expression was an independent indicator for overall survival in HCC patients (P < 0.05). CONCLUSIONS: The pathogenesis and progression of HCC are associated with reduced autophagy. The expression of Beclin-1 and Bax in HCC tissues may provide a synergistic effect towards inhibiting HCC proliferation, infiltration, metastasis and angiogenesis. Beclin-1 expression may be a valuable prognostic marker of HCC.

Study of RNA Interference Targeting NET-1 Combination with Sorafenib for Hepatocellular Carcinoma Therapy In Vitro and In Vivo.

The aim of this study is to explore the inhibitory effects of RNA interference (RNAi) targeting NET-1 or combined with sorafenib on HCC in vitro and in vivo and the possible underlying mechanisms. The expressions of NET-1 mRNA and protein were detected by RT-QPCR and western blot. The ability of proliferation was determined by CCK-8 assay. Apoptosis was examined by flow cytometry (FCM). Abilities of migration and invasion were measured by scratch-wound assay and transwell assay. MHCC97H cells with stable transfection of NET-1shRNA were injected subcutaneously to prepare nude mice model of HCC and Caspase-3, Caspase-8, and Caspase-9 mRNAs of tumor tissues in different groups were examined. NET-1 mRNA and protein were reduced sharply in MHCC97H cells transfected with NET-1shRNA. The abilities of proliferation and migration were inhibited and apoptosis was promoted in either NET-1shRNA or sorafenib as compared with untreated cells in vitro and in vivo (P < 0.05). The mRNA levels of caspase-3, caspase-8, and caspase-9 of tumor tissues were reduced in different treatment groups compared with untreated group, particularly in combination group. (P < 0.05). The combination NET-1shRNA with sorafenib dramatically enhanced the effects of sorafenib antitumor ,which may involve in blocking ras signaling pathway and stimulating apoptotic pathways simultaneously.

A synthetic dsRNA, as a TLR3 pathwaysynergist, combined with sorafenib suppresses HCC in vitro and in vivo.

BACKGROUND: Recent studies have demonstrated that synthetic dsRNAs may produce therapeutic effects in a target-independent manner through stimulation of the toll-like receptor-3 (TLR3)/interferon pathway; as a result, angiogenesis and proliferation of tumor cells are inhibited. Thus, this pathway may become a potential target of dsRNA in tumor suppression. In this study, we evaluated the role of synthetic dsRNA as a TLR3 synergist and by combining with sorafenib in anti-hepatocellular carcinoma (HCC) in vitro and in vivo. METHODS: Four dsRNAs were designed and synthesized. One of them that was capable of activating TLR3 most effectively in human HCC cell line (HepG2.2.15) was selected as a TLR3 synergist (called BM-06). Subsequently, the expression of proteins relating to TLR3 signaling pathway, such as NF-κB, caspase 8 survivin, bcl-2 and PCNA affected by BM-06, sorafenib alone or in combination, was compared. The migration, proliferation and apoptosis of HepG2.2.15 cells were evaluated in presence of BM-06, sorafenib alone or in combination of both. The similar treatments were also applied in an SD rat primary HCC model. RESULTS: qRT-PCR data showed that the expression of TLR3 and NF-κB in HepG2.2.15 cells was enhanced. BM-06 was selected as a TLR3 synergist capable of activating the TLR3/interferon pathway most effective among 4 synthetic dsRNAs. The migration and proliferation were significantly inhibited in treated HepG2.2.15 cells with BM-06 or Sorafenib alone as compared with PBS-sham control (P<0.01). However, the role of combination BM-06 with Sorafenib was the most prominent. Tumor cell apoptotic rate was increased by BM-06 or combination when compared to PBS or poly(I:C) (P<0.05). Similarly, in orthotopic HCC SD rats, the effect of the combination was superior to either agent alone on the inhibition of tumor growth and induction of HCC cell apoptosis (P<0.05). CONCLUSIONS: dsRNA alone was capable of inhibiting the proliferation of HepG2.2.15 cells and tumor growth of orthotopic HCC SD rats, but the effect of combination of dsRNA with sorafenib was more prominent. These findings implicate the potential role of combined use of a dsRNA, a TLR3 synergist, and sorafenib in inhibition of HCC.

Inhibitory effect of dsRNA TLR3 agonist in a rat hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumor. Studies have demonstrated that the toll­like receptor 3 (TLR3)/interferon pathway is inhibitory in cancer cell proliferation, suggesting that the activation of this pathway may have therapeutic potential. In the present study, the inhibitory effects of BM­06, a double­stranded (ds)RNA TLR3 agonist, against HCC were studied in vivo. Using a 2­acetylaminofluorene-induced HCC rat model, histological examination and analysis of corresponding biomarkers following treatment with BM-06, showed a decrease in tumor growth and cell proliferation, and an increase in apoptosis compared with that in a phosphate­buffered saline control group. In addition, the observed antitumor effect of BM­06 in the HCC rat model was demonstrated to be superior to the known TLR3 agonist, polyinosinic-polycytidylic acid.

TLR3 dsRNA agonist inhibits growth and invasion of HepG2.2.15 HCC cells.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). TLR3 expression and function in cancer cells are not well understood. In this study, we investigated whether TLR3 agonist dsRNA (BM-06) can inhibit proliferation and invasion, and promote apoptosis in HepG2.2.15 cells. HepG2.2.15 cells secreting hepatitis B virus (HBV) were treated with BM-06 and poly(I:C). Western blot analysis and PCR were employed to determine pharmacodynamic changes in biomarkers relevant to TLR3 signaling. Cell proliferation, invasion and apoptosis were analyzed by CCK-8 assay, transwell assay and flow cytometry. The expression of HBsAg, and HBcAg was observed by immunohistochemistry. Compared with untreated cells, pharmacological NF-κB activity of the TLR3 pathway by BM-06 (1.734-fold) or poly(I:C) (1.377-fold) was induced. By western blot analysis, we found that dsRNA induced TLR3-activated HepG2.2.15 cells which expressed NF-κB levels predominantly in the cytoplasmic fraction but fewer signals in the nucleus. BM-06 inhibited the proliferation, invasion and secretion of HBV, and induced apoptosis in HepG2.2.15 cells. In addition, the antitumor effects of BM-06 were superior to poly(I:C). Pharmacological activation of the TLR3 pathway by BM-06 can inhibit HepG2.2.15 cell growth.