[Clinical Value of Peripheral Blood Circulating Tumor Cells Detection in Early Cervical Infiltrating Carcinoma Screening].

OBJECTIVE: To observe the difference of circulating tumor cells (CTC) in peripheral blood of patients with different degrees of cervical lesions, and to evaluate the effectiveness of CTC detection in screening early invasive cervical cancer. METHODS: From December 2015 to October 2017, 63 cases of cervicitis, low-grade and high-grade intraepithelial lesions (LSIL, HSIL) and early invasive cervical cancer were confirmed by histopathological and clinical stages in Zhongshan Boai Hospital and Zhongshan Hospital Affiliated to Zhongshan University. The immunomagnetic bead negative enrichment technique combined with immunofluorescence was used. In situ hybridization (imFISH) was used to detect CTC in peripheral blood of patients. The positive rate and quantity of CTC in four groups were analyzed. The diagnostic efficacy of CTC in early invasive cervical cancer was evaluated based on the results of histopathological diagnosis and clinical staging. RESULTS: ①The positive rates of CTC in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group were 0, 0, 19.05% and 84.13% respectively. The overall difference was statistically significant ( χ 2=504.00， P<0.05). The positive rate of CTC in early invasive cervical cancer group was higher than that in other groups ( P<0.008 3). The positive rates of CTC in HSIL group were significantly different from those in LSIL group and cervicitis group ( P<0.008 3). ②The average number (median) of CTC positive in cervicitis group, LSIL group, HSIL group and early invasive cervical cancer group was 0, 0, 1/4 mL, 3/4 mL, respectively. The average number of positive CTC in early invasive cervical cancer group was higher than that in other groups, and the difference was significant compared with other groups ( P<0.008 3). The average number of CTC positive in HSIL group was significantly different from that in LSIL and cervicitis group ( P<0.008 3). ③The sensitivity, specificity, coincidence rate, positive predictive value and negative predictive value of CTC positive results in the diagnosis of early invasive cervical cancer were 84.13%, 93.65%, 91.27%, 81.54% and 94.65%, respectively. CONCLUSION: CTC exists in patients with HSIL and early invasive cervical cancer. With the aggravation of cervical lesions, the positive rate and number of CTC test results increase. CTC detection in early invasive cervical cancer screening has a certain practical value in clinic.

ZBP-89 and Sp1 contribute to Bak expression in hepatocellular carcinoma cells.

BACKGROUND: Kruppel family member zinc binding protein 89 (ZBP-89), also known as ZNF148, regulates Bak expression via binding to GC-rich promoter domain. It is not clear if other GC-rich binding factors, such as Sp family members, can interact with ZBPp-89 on Bak expression. This study aims to elucidate the mechanism of Bak expression regulation by ZBP-89 and Sp proteins, based on in vitro experiment and The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data cohort. METHODS: We downloaded TCGA hepatocellular carcinoma (HCC) cohort data to analysis the association of Bak transcription level with ZBP-89 and Sp proteins transcription level. HCC cell lines and liver immortal non-tumour cell lines were used for mechanism study, including western blotting analysis, expression vector mediated gene expression and siRNA interference. RESULTS: Results showed that cancer tissues have higher Bak transcription level compared with adjacent non-cancer tissues. Bak transcription level was correlated with Sp1 and Sp3 expression level, while no correlation was found in ZBP-89 and Bak, neither Sp2 nor Sp4. Mithramycin A (MMA) induced Bak expression in a dose-dependent manner. Western blotting results showed Sp1 overexpression increased Bak expression both in liver immortal non-tumour cells and HCC cells. Interference Sp1 expression could inhibit Bak expression alone. ZBP-89 siRNA suppressed Bak expression even in the presence of MMA treatment and S1 overexpression. Additionally, Bak and Sp1 level were associated with HCC patient survival. CONCLUSIONS: Bak expression required ZBP-89 and Sp1 cooperative regulation simultaneously.

ZBP-89 reduces histone deacetylase 3 by degrading IkappaB in the presence of Pin1.

BACKGROUND: Histone deacetylase 3 (HDAC3) is overexpressed in cancers and its inhibition enhances anti-tumor chemotherapy. ZBP-89, a transcription factor, can induce pro-apoptotic Bak and reduce HDAC3 but the mechanism is unknown. Pin1, a molecular switch that determines the fate of phosphoproteins, is known to interact with HDAC3. The aim of this study was to investigate the mechanism how ZBP-89 downregulated HDAC3. METHODS: In this study, liver cells, Pin1-knockout Pin1(-/-) and Pin1 wild-typed Pin(+/+) cells were used to explore how ZBP-89 reduced HDAC3. The overexpression of ZBP-89 was achieved by infecting cells with Ad-ZBP-89, an adenoviral construct containing ZBP-89 gene. The role of NF-κB was determined using CAY10576, MG132 and SN50, the former two being inhibitors of IκB degradation and SN50 being an inhibitor of p65/p50 translocation. A xenograft tumor model was used to confirm the in vitro data. RESULTS: ZBP-89 reduced HDAC3, and it could form a complex with IκB and induce IκB phosphorylation to inhibit IκB. Furthermore, ZBP-89-mediated HDAC3 reduction was suppressed by IκB degradation inhibitors CAY10576 and MG132 but not by p65/p50 translocation inhibitor SN50, indicating that IκB decrease rather than the elevated activity of NF-κB contributed to HDAC3 reduction. ZBP-89-mediated HDAC3 or IκB reduction was significantly less obvious in Pin1(-/-) cells compared with Pin1(+/+) cells. In Ad-ZBP-89-infected Pin1(+/+) cancer cells, Pin1 siRNA increased HDAC3 but decreased Bak, compared with cells without ZBP-89 infection. These findings indicate that Pin1 participates in ZBP-89-mediated HDAC3 downregulation and Bak upregulation. The cell culture result was confirmed by in vivo mouse tumor model experiments. CONCLUSIONS: ZBP-89 attenuates HDAC3 by increasing IκB degradation. Such attenuation is independent of NF-κB activity but partially depends on Pin1. The novel pathway identified may help generate new anti-cancer strategy by targeting HDAC3 and its related molecules.

Therapeutic efficacy of improved α-fetoprotein promoter-mediated tBid delivered by folate-PEI600-cyclodextrin nanopolymer vector in hepatocellular carcinoma.

SNPs in human AFP promoter are associated with serum AFP levels in hepatocellular carcinoma (HCC), suggesting that AFP promoter variants may generate better transcriptional activities while retaining high specificity to AFP-producing cells. We sequenced human AFP promoters, cloned 15 different genotype promoters and tested their reporter activities in AFP-producing and non-producing cells. Among various AFP variant fragments tested, EA4D exhibited the highest reporter activity and thus was selected for the further study. EA4D was fused with tBid and coupled with nano-particle vector (H1) to form pGL3-EA4D-tBid/H1. pGL3-EA4D-tBid/H1 could express a high level of tBid while retain the specificity to AFP-producing cells. In a HCC tumor model, application of pGL3-EA4D-tBid/H1 significantly inhibited the growth of AFP-producing-implanted tumors with minimal side-effects, but had no effect on non-AFP-producing tumors. Furthermore, pGL3-EA4D-tBid/H1 could significantly sensitize HCC cells to sorafenib, an approved anti-HCC agent. Collectively, pGL3-EA4D-tBid/H1, a construct with the AFP promoter EA4D and the novel H1 delivery system, can specifically target and effectively suppress the AFP-producing HCC. This new therapeutic tool shows little toxicity in vitro and in vivo and it should thus be safe for further clinical tests.

Epigenetic upregulation of Bak by ZBP-89 inhibits the growth of hepatocellular carcinoma.

Zinc-binding protein-89 regulates Bak to facilitate apoptosis in cancer cells. This study examined if zinc-binding protein-89 regulates Bak through an epigenetic mechanism in hepatocellular carcinoma. We first demonstrated that the expression of Bak was reduced but the levels of deoxyribonucleic acid methyltransferase 1 and histone deacetylase 3 were increased in hepatocellular carcinoma cancer tissues compared to the corresponding non-cancer tissues. Moreover, there was a negative correlation between Bak expression and deoxyribonucleic acid methyltransferase 1 levels in hepatocellular carcinoma. Administration of zinc-binding protein-89 downregulated histone deacetylase 3 expression and suppressed the activities of histone deacetylase and deoxyribonucleic acid methyltransferase, which led to maintenance of histone acetylation status, inhibited the binding of methyl-CpG-binding protein 2 to genomic deoxyribonucleic acid and demethylated CpG islands in the Bak promoter in hepatocellular carcinoma cells. Using the xenograft mouse tumor model, we demonstrated that zinc-binding protein-89 or inhibitors of either epigenetic enzymes could stimulate Bak expression, induce apoptosis, and arrest tumor growth and that the maximal effort was achieved when zinc-binding protein-89 and the enzyme inhibitors were used in combination. Conclusively, zinc-binding protein-89 upregulates the expression of Bak by targeting multiple components of the epigenetic pathway in hepatocellular carcinoma.

Increased glutathione and mitogen-activated protein kinase phosphorylation are involved in the induction of doxorubicin resistance in hepatocellular carcinoma cells.

AIM: The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. METHODS: In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. RESULTS: Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. CONCLUSION: These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Baicalin attenuates TNBS-induced colitis in rats by modulating the Th17/Treg paradigm.

Baicalin, a flavonoid, has a wide range of pharmacological properties, including immunomodulation. The objective of this study was to investigate the effect of baicalin on the balance of T helper 17 (Th17) and regulatory T (Treg) cells in a colitis model. The rat colitis model was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Baicalin (10 ml/kg, each) or mesalazine (positive control) was then administered orally for 7 days. Inflammatory and immunological responses were evaluated by pathology, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, western blot analysis, and flow cytometry. Our study showed that baicalin not only significantly attenuated TNBS-induced colitis by reducing the disease activity index as well as macroscopic and microscopic scores, but it also improved the weight loss and shortening of the colon. Baicalin treatment also induced a significant decrease in the levels of inflammatory mediators, including the myeloperoxidase activity, the levels of tumor necrosis factor α, IL-1ß, and Th1-related cytokines IL-12 and IFN-γ. Furthermore, the beneficial effects of baicalin seem to be associated with regulation of the Th17 and Treg paradigm. We found that administration of baicalin significantly downregulated the number of Th17 cells and the levels of Th17-related cytokines (IL-17 and IL-6) and retinoic acid receptor-related orphan receptor γt. In contrast, there was an increase in Treg cells numbers, Treg-related cytokines transforming growth factor-ß and IL-10, and forkhead box P3. Our results suggest that the anti-inflammatory effect of baicalin may be linked to modulation of the balance between Th17 and Treg cells in TNBS-induced ulcerative colitis.

Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression.

Doxorubicin is a chemotherapeutic drug widely used for the treatment of hepatocellular carcinoma but its efficacy is restricted by multidrug resistance. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors exhibit anti-cancer properties as well as abilities to overcome drug resistance. In the present study, indomethacin (a NSAID) and SC236 (a COX-2-selective inhibitor) enhanced the cytotoxicity of doxorubicin in the hepatocellular carcinoma cell line HepG2 and its drug-resistant sub-line R-HepG2. Both drugs increased the intracellular accumulation and retention of doxorubicin in vitro. The effects were not reversed by prostaglandin E(2), implicating a COX-independent mechanism. Indomethacin and SC236 partially reversed the increase in expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) induced by doxorubicin in R-HepG2 cells. In conclusion, indomethacin and SC236 increased the intracellular accumulation and retention of doxorubicin and thus its cytotoxicity in HepG2 and drug-resistant HepG2 cells. These effects, mediated through decrease in P-gp and MRP1 expression and/or direct inhibition of P-gp activity, may improve multidrug resistant-cancer chemotherapy.

2,3',4,4',5'-Pentamethoxy-trans-stilbene, a resveratrol derivative, inhibits colitis-associated colorectal carcinogenesis in mice.

BACKGROUND AND PURPOSE: Resveratrol, a naturally occurring polyphenolic antioxidant, has been shown to exhibit chemoprophylactic effects on cancer development. Previously, we reported that 2,3',4,4',5'-pentamethoxy-trans-stilbene (PMS), a methoxylated resveratrol derivative, exerted a highly potent anti-proliferative effect on human colon cancer cells as compared with its parent compound. In the present study, the chemopreventive effect of PMS was evaluated in a mouse model of colitis-associated colon carcinogenesis. EXPERIMENTAL APPROACH: Seven-week-old Balb/c mice were injected i.p. with 10 mg.kg(-1) azoxymethane (AOM). After 1 week, 3% dextran sodium sulphate (DSS) was administered in the drinking water for 7 days followed by 14 days of tap water for recovery, and this cycle was repeated twice. KEY RESULTS: Intragastric administration of PMS (25, 50 mg.kg(-1) body weight) for 16 weeks significantly reduced the multiplicity of colonic neoplasms by 15% and 35% (P < 0.01) respectively. Moreover, PMS at 50 mg.kg(-1) inhibited colon cancer cell proliferation and promoted apoptosis. Such changes were accompanied by reduction of Akt (protein kinase B) phosphorylation, inactivation of beta-catenin and down-regulation of inducible nitric oxide synthase. In parallel, in vitro studies also demonstrated that PMS inhibited proliferation and induced apoptosis in the murine colon adenocarcinoma cell line Colon26 with concomitant inhibition of Akt phosphorylation and inactivation of beta-catenin. CONCLUSIONS AND IMPLICATIONS: PMS effectively suppressed colon carcinogenesis in an AOM/DSS animal model and may merit further clinical investigation as a chemoprophylactic agent against colitis-associated colon cancer in humans.

3,3',4,5,5'-Pentahydroxy-trans-stilbene, a resveratrol derivative, induces apoptosis in colorectal carcinoma cells via oxidative stress.

Resveratrol exhibits anti-tumor properties against different types of cancer. In this study, several polyhydroxylated resveratrol derivatives were prepared with the aim of discovering new leading compounds with clinical potential for human colon cancer chemotherapy. Among these compounds, 3,3',4,5,5'-pentahydroxy-trans-stilbene (PHS) displayed the most potent cytotoxicity and triggered apoptosis in HT-29 cells as evidenced by increased poly(ADP-ribose) polymerase (PARP) cleavage, elevated levels of cytoplasmic nucleosomes and DNA fragmentation. Further mechanistic analysis revealed that PHS-induced apoptosis was caspase-dependent and mediated by its pro-oxidative action through up-regulation of reactive oxidative species generation and depletion of intracellular glutathione.