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Fulvestrant-3-boronic acid (ZB716), an oral selective estrogen receptor degrader (SERD) under clinical development, has been investigated in ADME studies to characterize its absorption, metabolism, and pharmacokinetics. ZB716 was found to have high plasma protein binding in human and animal plasma, and low intestinal mucosal permeability. ZB716 had high clearance in hepatocytes of all species tested. ZB716 was metabolized primarily by CYP2D6 and CYP3A. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). In assays for enzyme activity, ZB716 induced CYP1A2, 2B6, and 3A4 in a concentration-dependent manner. Single-dose and repeated-dose pharmacokinetic studies in rats and dogs showed oral bioavailability, dose-proportional drug exposure, and drug accumulation as measured by maximum concentration and area under the concentration–time curve (AUC).

The purpose of this study was to identify genotypes associated with dose-adjusted tacrolimus trough concentrations (C0/D) in kidney transplant recipients using whole-exome sequencing (WES). This study included 147 patients administered tacrolimus, including seventy-five patients in the discovery set and seventy-two patients in the replication set. The patient genomes in the discovery set were sequenced using WES. Also, known tacrolimus pharmacokinetics-related intron variants were genotyped. Tacrolimus C0/D was log-transformed. Sixteen variants were identified including novel CYP3A7 rs12360 and rs10211 by ANOVA. CYP3A7 rs2257401 was found to be the most significant variant among the periods by ANOVA. Seven variants including CYP3A7 rs2257401, rs12360, and rs10211 were analyzed by SNaPshot in the replication set and the effects on tacrolimus C0/D were verified. A linear mixed model (LMM) was further performed to account for the effects of the variants and clinical factors. The combined set LMM showed that only CYP3A7 rs2257401 was associated with tacrolimus C0/D after adjusting for patient age, albumin, and creatinine. The CYP3A7 rs2257401 genotype variant showed a significant difference on the tacrolimus C0/D in those expressing CYP3A5, showing its own effect. The results suggest that CYP3A7 rs2257401 may serve as a significant genetic marker for tacrolimus pharmacokinetics in kidney transplantation.

Human papillomaviruses (HPVs) are small DNA viruses; some oncogenic ones can cause different types of cancer, in particular cervical cancer. HPV-associated carcinogenesis provides a classical model system for RNA interference (RNAi) based cancer therapies, because the viral oncogenes E6 and E7 that cause cervical cancer are expressed only in cancerous cells. Previous studies on the development of therapeutic RNAi facilitated the advancement of therapeutic siRNAs and demonstrated its versatility by siRNA-mediated depletion of single or multiple cellular/viral targets. Sequence-specific gene silencing using RNAi shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, siRNA-based targeting requires further validation of its efficacy in vitro and in vivo, for its potential off-target effects, and of the design of conventional therapies to be used in combination with siRNAs and their drug delivery vehicles. In this review we discuss what is currently known about HPV-associated carcinogenesis and the potential for combining siRNA with other treatment strategies for the development of future therapies. Finally, we present our assessment of the most promising path to the development of RNAi therapeutic strategies for clinical settings.

Nanodiamonds have been discovered as a new exogenous material source in biomedical applications. As a new potent form of nanodiamond (ND), polyamidoamine-decorated nanodiamonds (PAMAM-NDs) were prepared for E7 or E6 oncoprotein-suppressing siRNA gene delivery for high risk human papillomavirus-induced cervical cancer, such as types 16 and 18. It is critical to understand the physicochemical properties of siRNA complexes immobilized on cationic solid ND surfaces in the aspect of biomolecular structural and conformational changes, as the new inert carbon material can be extended into the application of a gene delivery vector. A spectral study of siRNA/PAMAM-ND complexes using differential scanning calorimetry and circular dichroism spectroscopy proved that the hydrogen bonding and electrostatic interactions between siRNA and PAMAM-NDs decreased endothermic heat capacity. Moreover, siRNA/PAMAM-ND complexes showed low cell cytotoxicity and significant suppressing effects for forward target E6 and E7 oncogenic genes, proving functional and therapeutic efficacy. The cellular uptake of siRNA/PAMAM-ND complexes at 8 h was visualized by macropinocytes and direct endosomal escape of the siRNA/PAMAM-ND complexes. It is presumed that PAMAM-NDs provided a buffering cushion to adjust the pH and hard mechanical stress to escape endosomes. siRNA/PAMAM-ND complexes provide a potential organic/inorganic hybrid material source for gene delivery carriers.

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Introduction Response to mesenchymal-epithelial transition (MET) inhibitors in NSCLC with mesenchymal-epithelial transition gene ( MET ) exon 14 skipping ( MET ex14) has fueled molecular screening efforts and the search for optimal therapies. However, further work is needed to refine the clinicopathologic and prognostic implications of MET ex14 skipping. Methods Among 795 East Asian patients who underwent a surgical procedure for NSCLC, we screened 45 patients with quintuple-negative ( EGFR -negative /KRAS -negative / anaplastic lymphoma kinase gene [ ALK ]-negative /ROS1 -negative / ret proto-oncogene [ RET ]-negative) lung adenocarcinomas by using reverse-transcriptase polymerase chain reaction and found 17 patients (37.8%) with MET ex14 skipping. We also investigated the effect of small interfering RNA (siRNA) targeting skipping junction in cells with MET ex14 skipping. Results The median age of the 17 patients was 73 years. The acinar subtype was predominant (52.9%), followed by the solid subtype (35.3%). MET immunohistochemistry demonstrated 100% sensitivity and 70.4% specificity. Multivariate analyses showed that patients with MET ex14 skipping had a higher recurrence rate than those with ALK fusion (versus MET ex14 skipping) (hazard ratio = 0.283, 95% confidence interval: 0.119 – 0.670) in stage I to IIIA disease; however, the differences in overall survival were not significant after adjustment for pathologic stage ( p = 0.669). Meanwhile, siRNA decreased MET-driven signaling pathways in Hs746T cells, and combined treatment with siRNA and crizotinib inhibited cell proliferation in crizotinib-resistant H596 cells. Conclusions The prevalence of MET ex14 skipping was quite high in East Asian patients without other driver mutations in lung adenocarcinomas. MET ex14 skipping was associated with old age, the acinar or solid histologic subtype, and high MET immunohistochemical expression. The prognosis of patients with MET ex14 skipping was similar to that of patients with major driver mutations. siRNA targeting the junction of MET ex14 skipping could inhibit MET-driven signaling pathways in cells with MET ex14 skipping.

The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.

Unlike epigenetic silencing of tumor suppressor genes, the role of epigenetic derepression of cancer-promoting genes or oncogenes in carcinogenesis remains less well understood. The tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer and their overexpression was previously reported to promote the migration and invasion of ovarian epithelial cells. Here, we show that the expression of claudin-3 and claudin-4 is repressed in ovarian epithelial cells in association with promoter 'bivalent' histone modifications, containing both the activating trimethylated histone H3 lysine 4 (H3K4me3) mark and the repressive mark of trimethylated histone H3 lysine 27 (H3K27me3). During ovarian tumorigenesis, derepression of CLDN3 and CLDN4 expression correlates with loss of H3K27me3 in addition to trimethylated histone H4 lysine 20 (H4K20me3), another repressive histone modification. Although CLDN4 repression was accompanied by both DNA hypermethylation and repressive histone modifications, DNA methylation was not required for CLDN3 repression in immortalized ovarian epithelial cells. Moreover, activation of both CLDN3 and CLDN4 in ovarian cancer cells was associated with simultaneous changes in multiple histone modifications, whereas H3K27me3 loss alone was insufficient for their derepression. CLDN4 repression was robustly reversed by combined treatment targeting both DNA demethylation and histone acetylation. Our study strongly suggests that in addition to the well-known chromatin-associated silencing of tumor suppressor genes, epigenetic derepression by the conversely related loss of repressive chromatin modifications also contributes to ovarian tumorigenesis via activation of cancer-promoting genes or candidate oncogenes.

Kochujang (Korean fermented red pepper paste) is a mixture of fermented soybeans, wheat, and red pepper powder. Kochujang has been reported to reduce body fat gain and lipid levels of adipose tissues and serum in rats. We studied the inhibitory effect of Kochujang on lipid accumulation and investigated the molecular mechanism of the action in 3T3-L1 adipocytes by measuring the expression levels of adipocyte-specific genes by real-time reverse transcription-polymerase chain reaction. When 3T3-L1 adipocytes were treated with Kochujang extract (KE), the sizes of adipocytes and leptin secretion were decreased. Hormone-sensitive lipase (HSL) was transcriptionally up-regulated at 4 hours, and glycerol secretion was increased at both 4 hours and 24 hours. Moreover, mRNA expression levels of both sterol regulatory element-binding protein 1-c (SREBP-1c) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which are critical transcription factors for adipogenesis, were markedly down-regulated. Tumor necrosis factor-alpha (TNF-alpha) is reported to impair pre-adipocyte differentiation and induce lipolysis and apoptosis. KE treatment of 3T3-L1 adipocytes decreased TNF-alpha mRNA levels, but had no apparent affect on apoptosis. Taken together, our study shows that Kochujang decreased lipid accumulation in 3T3-L1 adipocytes by inhibiting adipogenesis through down-regulation of SREBP-1c and PPAR-gamma and by stimulation of lipolysis due to increased HSL activity. TNF-alpha might not be involved in the reduction of lipid accumulation by KE.

The hedgehog (Hh)-signaling pathway plays an essential role in normal development. Deregulation of this pathway is responsible for several types of cancers. The aim of this study was to determine the expression pattern and the extent of Hh-signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions. A total of 106 uterine cervical cancers and related lesions (37 squamous cell carcinomas, 23 cervical intraepithelial neoplasia (CIN) III, 10 CIN II, four CIN I, 32 normal cervical epithelia) were immunohistochemically analyzed with anti-Shh, Indian Hh (Ihh), Patched (PTCH), Smoothened (Smo), Gli-1, Gli-2, Gli-3 antibodies on paraffin blocks. The results showed that the expression of all the Hh-signaling molecules was greatly enhanced in uterine cervical tumors, including carcinoma and its precursor lesions. The staining pattern was mainly cytoplasmic except for Gli-1/2, whose expression was observed in both cytoplasm and nucleus. In case of Ihh, PTCH, Smo and Gli-1, their expression in normal epithelium was completely absent or rare. The expression of all the seven Hh-signaling molecules mentioned above was significantly increased in CIN II/III and carcinoma, compared with that in normal epithelium (P < 0.05). The expression of Shh was increased by double; the first increase occurred in normal epithelium-CIN transition, and the second, during the progression of CIN to carcinoma. These results strongly suggest that the Hh-signaling pathways were extensively activated in carcinoma and CIN of uterine cervix. In conclusion, the Hh-signaling pathways may be involved in carcinogenesis of squamous cell carcinoma of uterine cervix and can be considered as a potential therapeutic target.

To elucidate the effects of retinoic acids (RAs) on adipogenesis and insulin sensitivity, we treated mature adipocytes with two different kinds of RA, 9-cis-RA and all-trans-RA. Both 9-cis- and all-trans-RA inhibited the secretion of leptin. However, the inhibition was significantly decreased at a higher dose of each RA. The inhibitory effect of 9-cis-RA was synergistically enhanced by the addition of rosiglitazone, a synthetic ligand for peroxisome proliferator-activated receptor (PPAR) gamma. 9-cis-RA also leads to adipogenesis in a dose-dependent manner. On the contrary, all-trans-RA does not increase adipogenesis in a dose-dependent manner. To clarify the antidiabetic effects of RA, glucose uptake was assessed by estimating glucose concentrations in the medium. 9-cis-RA reduced glucose levels in the culture media, but all-trans-RA did not. In conclusion, all-trans-RA does not alter adipogenesis and glucose uptake but does inhibit leptin secretion. 9-cis-RA, however, seems to increase both adipogenesis and glucose uptake through activation of the retinoid X receptor/PPARgamma heterodimer.

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA, the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Human papillomavirus (HPV) E6 and E7 viral oncogenes are very well known to cause cervical cancer, because E6 degrades TP53 tumor suppressor protein, and E7 inactivates the tumor suppressor retinoblastoma (pRb) protein. Thus E6 and E7 oncogenes of HPV are supposed to be promising targets of gene therapy against HPV mediated cervical cancer. Here, we attempted to study the regulation of TP53/pRb proteins dynamic behaviour after HPV E6/E7 small interfering RNA (siRNA) transfection in cervical cancer cells. HPV positive (HeLa and Caski) cell lines were selected for these experiments. Herein, we also validated the dynamics of TP53 in response to antiviral siRNA targeting the E6 and E7 oncogenes. At first, we analyzed the effect of HPV E6/E7 siRNA on TP53/pRb reporter gene activity by using pathway profiling luciferase system. Our result clearly indicates that when compared with E6/E7 siRNA pool alone, siRNA pool in combination cisplatin dramatically increases the TP53/pRb and decreases E2F response elements regulating luciferase reporter gene activity. TP53 protein and its target gene expression were also confirmed by western blot and qRT-PCR analysis in a time dependent manner. In our TP53 reporter gene (pGreenFire1 GFP reporter vector with EF1-puro) assay by using live imaging analysis shows that combination of HPVE6/E7 siRNA pool with cisplatin, induces the TP53 expression within 10h and sustained that leads to cell death. Our imaging results also suggested that combining cisplatin with inhibition of HPV E6/E7 oncogene would synergize to stimulate a sustained GFP-TP53 expression can trigger either cell cycle arrest or apoptosis in response to DNA damage. In addition, we generated GFP-TP53 reporter stable HeLa cell line (pGreenFire1 GFP reporter vector with EF1-puro), in order to find the specificity of HPV E6/E7 siRNA induced TP53 expression in in vitro and in vivo. Similar GFP-TP53 dynamic pattern was observed in E6/E7 siRNA transfected GFP-TP53 reporter stable HeLa cell line. Furthermore to analyze the dynamic behaviour of pRb/E2F in live cells, reporter vector containing luciferase gene was recombinantly engineered to replace with RFP gene. Taken together, our results suggest that increase of TP53/Rb expression by silencing E6/E7 is associated with its chemo-sensitizing effect in cervical cancer cells. Overall, stabilization and maximal activation of the TP53/pRb signaling network can facilitate determination of the optimal therapeutic strategy for human cervical carcinoma. . Citation Format: Nirmal Rajasekaran, Hun Soon Jung, Young Deug Kim, Deuk Ae Kim, Tae Kyung Ha, Yoo Ha Na, Young kee Shin. Effect of small interfering RNA targeting HPV E6/E7 gene on the regulation of TP53/Rb dynamic behaviour in cervical cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2115. doi:10.1158/1538-7445.AM2015-2115

1-8D gene is a member of human 1-8 interferon inducible gene family and was shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from human 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy, of three peptides we established an active immunization model using HHD mice. D b-/- ×β2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/D b- 2m single chain (HHD mice) were challenged with B16/HHD/1-8D tumor cells and were immunized with irradiated peptide-loaded RMA- S/HHD/B7.1 transfectants. In therapy model tumor growth was retarded in HHD mice that were injected with 3-5 peptide-loaded RMA-S/HHD/B7.1. In survival test vaccination with 1-8D-derived peptide protects HHD mice from tumor progression after tumor challenge. Results: These studies show that peptide 3-5 derived from 1-8D gene can be the most effective candidate for the vaccine of immunotherapy against colon cancer and highlight 1-8D gene as putative colon carcinoma associated antigens. Conclusion: We demonstrated that RMA-S/HHD/ B7.1 loaded with 1-8D peptides, especially 3-5, immunization generates potent antitumor immunity against tumor cells in HHD mice and designed active immunization as proper immunotherapeutic protocols.