Synergistic Effect of Ginsenoside Rh2 Combines with Ionizing Radiation on CT26/luc Colon Carcinoma Cells and Tumor-Bearing Animal Model.

BACKGROUND: The local tumor control rate of colon cancer by radiotherapy is unsatisfactory due to recurrence and radioresistance. Ginsenoside Rh2 (Rh2), a panoxadiol saponin, possesses various antitumor effects. METHODS: CT26/luc murine colon carcinoma cells and a CT26/luc tumor-bearing animal model were used to investigate the therapeutic efficacy of Rh2 combined with ionizing radiation and the underlying mechanisms. RESULTS: Rh2 caused cell cycle arrest at the G1 phase in CT26/luc cells; however, when combined with ionizing radiation, the cells were arrested at the G2/M phase. Rh2 was found to suppress the activity of NF-κB induced by radiation by inhibiting the MAPK pathway, consequently affecting the expression of effector proteins. In an in vivo study, the combination treatment significantly increased tumor growth delay time and overall survival. Furthermore, the combination treatment significantly reduced NF-κB and NF-κB-related effector proteins, along with PD-1 receptor expression. Additionally, Rh2 administration led to increased levels of interleukin-12, -18, and interferon-γ in the mice's sera. Importantly, biochemical analysis revealed no toxicities associated with Rh2 alone or combined with radiation. CONCLUSIONS: The combination of Rh2 with radiation may have potential as an alternative to improve the therapeutic efficacy of colorectal cancer.

The effects of patchouli alcohol and combination with cisplatin on proliferation, apoptosis and migration in B16F10 melanoma cells.

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G0 /G1 phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.

Cedrol restricts the growth of colorectal cancer in vitro and in vivo by inducing cell cycle arrest and caspase-dependent apoptotic cell death.

Background: Cedrol is a natural sesquiterpene alcohol found in Cedrus atlantica, which has been proven to have a broad spectrum of biological activities, such as antimicrobial, anti-inflammatory, analgesic, anxiolytic, and anti-cancer effects. However, the underlying anticancer mechanisms and in vivo inhibitory effects of cedrol on colorectal cancer (CRC) have not been elucidated. In the present study, we investigated the anti-CRC potential of cedrol using in vitro and in vivo models. Methods: The effects of cedrol on cell viability, cell cycle progression, and apoptosis of HT-29 and CT-26 cells were detected by MTT, flow cytometry, and TUNEL assays. Western blotting was used to measure protein expression for molecular signaling analyses. Results: Cedrol inhibited HT-29 and CT-26 cell proliferation in a time- and dose-dependent manner, with IC50 values of 138.91 and 92.46 µM, respectively. Furthermore, cedrol induced cell cycle arrest at the G0/G1 phase by regulating the expression of cell cycle regulators, such as CDK4 and cyclin D1, and triggered apoptosis through extrinsic (FasL/caspase-8) and intrinsic (Bax/caspase-9) pathways. In addition, cedrol in combination with the clinical drug 5-fluorouracil exhibited synergistic inhibitory effects on CRC cell growth. Importantly, cedrol treatment suppressed the progression of CRC and improved the survival rate of animals at a well-tolerated dose. Conclusion: These results suggest that cedrol has an anti-cancer potential via induction of cell cycle arrest and apoptosis, and it could be considered as an effective agent for CRC therapy.

Risk of uterine leiomyoma based on BET1L rs2280543 single nucleotide polymorphism and vegetarian diet.

BACKGROUND: Bet1 Golgi vesicular membrane trafficking protein-like (BET1L) rs2280543 single nucleotide polymorphism (SNP) and diet have been independently associated with uterine leiomyoma (UL). However, whether the SNP and diet could jointly influence the risk of UL is yet to be assessed. Therefore, we investigated the independent and interactive effects of vegetarian diet and BET1L rs2280543 on uterine fibroids in Taiwanese women. METHODS: We linked participants' electronic data in the Taiwan Biobank (TWB) database to their medical records in the National Health Insurance Research Database (NHIRD). The TWB had genotypic, lifestyle, and biochemical data between 2008 and 2015 and the NHIRD had data on disease diagnoses between 1998 and 2015. In this study, we included 1997 premenopausal women with complete data. RESULTS: Compared  to participants with the BET1L rs2280543 CC genotype (wildtype), those with CT/CC genotype had an odds ratio (OR) of 0.69 and a 95% confidence interval (CI) of 0.51-0.93. Vegetarian diet and UL were not significantly associated: OR = 1.09 and 95% CI = 0.77-1.55. However, the test for interaction between rs2280543 and vegetarian diet was significant (p = 0.046). Compared to individuals with the CC genotype, the risk of UL was lower among vegetarians with the CT/TT genotype: OR (95% CI) = 0.15 (0.05-0.47). CONCLUSION: The BET1L rs2280543 CT/TT genotype was associated with a lower risk of UL especially among vegetarians.

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo.

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Evaluation of anticancer effects of Juniperus communis extract on hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and accounts for the fourth leading cause of all cancer deaths. Scientific evidence has found that plant extracts seem to be a reliable choice due to their multitarget effects against HCC. Juniperus communis has been used for centuries in traditional medicine and its anticancer properties have been reported. As a result, the purpose of the study was to investigate the anticancer effect and mechanism of J. communis extract (JCo extract) on HCC in vitro and in vivo. In the present study, we found that JCo extract inhibited the growth of human HCC cells by inducing cell cycle arrest at the G0/G1 phase, extensive apoptosis and suppressing metastatic protein expressions in HCC cells. Moreover, the combinational treatment of JCo and VP-16 was found to enhance the anticancer effect, revealing that JCo extract might have the potential to be utilized as an adjuvant to promote HCC treatment. Furthermore, in vivo study, JCo extract significantly suppressed HCC tumor growth and extended the lifespan with no or low systemic and pathological toxicity. JCo extract significantly up-regulated the expression of pro-apoptotic proteins and tumor suppressor p53, suppressed VEGF/VEGFR autocrine signaling, down-regulated cell cycle regulatory proteins and MMP2/MMP9 proteins. Overall, our results provide a basis for exploiting JCo extract as a potential anticancer agent against HCC.

Cell cycle arrest and apoptosis induction by Juniperus communis extract in esophageal squamous cell carcinoma through activation of p53-induced apoptosis pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers. It has a high mortality rate and requires novel effective drugs and therapeutic approaches. Juniperus communis (JCo), used to flavor gin and food, has been documented to have anti-tumor activity. The aim of this study was to investigate the antitumor activity of JCo extract against ESCC and its possible mechanisms. JCo extract suppressed cell growth in ESCC and showed higher selection for ESCC cells than normal cells compared to the clinical drug 5-fluorouracil (5-FU). JCo extract induced cell cycle arrest at the G0/G1 phase by regulating the expression of p53/p21 and CDKs/cyclins, triggering cell apoptosis by activating both the extrinsic (Fas/FasL/Caspase 8) and intrinsic (Bcl-2/Bax/Caspase 9) apoptosis pathways. Moreover, a combination treatment of JCo and 5-FU synergistically inhibited proliferation of ESCC cells. These results suggest that JCo extract is a potential natural therapeutic agent for esophageal cancer, as it could induce cell cycle arrest and apoptosis in ESCC cells.

Juniperus indica Bertol. extract synergized with cisplatin against melanoma cells via the suppression of AKT/mTOR and MAPK signaling and induction of cell apoptosis.

Juniperus indica Bertol. is an herbal plant that belongs to the genus Juniperus, which is commonly used in traditional medicine to refresh the mind and for diuretic use. However, few studies have reported the function of J. indica Bertol. Hence, this study aimed to investigate the anti-tumor and synergistic potential of J. indica Bertol. extract (JIB extract) for melanoma cells. Our results indicated the anti-melanoma activity of JIB extract. JIB extract induced cell cycle arrest at the G0/G1 phase and decreased cyclin and cdk protein expressions. In addition, AKT/mTOR signaling and MAPK signaling were inhibited by JIB extract to suppress melanoma cell growth and proliferation. Additionally, JIB extract induced B16/F10 cell apoptosis via the caspase cascade. According to the JIB extract's anti-melanoma capacity, to assess the synergistic effects of cisplatin and JIB extract. The results demonstrated that JIB extract combined with cisplatin enhanced the inhibition of cell growth, proliferation, and survival through the obstruction of cell cycle progression and AKT/mTOR and MAPK signaling as well as the induction of cell apoptosis. Collectively, our results indicate that JIB extract showed anti-tumor effects and synergized with cisplatin against B16/F10 cells, indicating the possibility of JIB extract to be developed as adjuvant therapy for melanoma.

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Effects of Cedrus atlantica extract on acute myeloid leukemia cell cycle distribution and apoptosis.

This study investigated the anti-leukemic effects of Cedrus atlantica extract (CAt extract) on cell cycle distribution and apoptosis in human acute myeloid leukemia (AML) cells. AML often occurs in older adults, accounting for 60% of the cases, and is likely to be resistant to chemotherapy due to multidrug resistance, resulting in early death during cancer treatment. With the increasing focus on prevention medicine, natural plant components are being used as a major source for the development of therapeutic drugs or functional foods to cure or alleviate the disease. Cedrus species are known to have anti-inflammatory, antimicrobial, antiviral, and anticancer effects; however, the anticancer effects of CAt extract have not been elucidated. In this study, CAt extract demonstrated an inhibitory effect on human leukemia cells in a concentration-dependent manner; CAt extract induced G0/G1 phase arrest via restrained protein levels of p-Rb and cell cycle-related proteins. After CAt extract exposure, the extrinsic and intrinsic apoptotic pathways were activated through caspase-8, -9, and -3 cleavage. Additionally, CAt extract suppressed VEGF, MMP-2, and MMP-9 expression. This study demonstrated that CAt extract treatment significantly reduced cell growth, cell cycle arrest in the G0/G1 phase, and induction of apoptosis, leading to leukemia cell death.

Extract Derived from Cedrus atlantica Acts as an Antitumor Agent on Hepatocellular Carcinoma Growth In Vitro and In Vivo.

Cedrus atlantica is widely used in herbal medicine. However, the anti-cancer activity of C. atlantica extract (CAt extract) has not been clarified in hepatocellular carcinoma. In the study, we elucidated the anti-hepatoma capacity of CAt extract on HCC in vitro and in vivo. To explore the anti-hepatoma mechanisms of the CAt extract in vitro, HCC and normal cells were treated with the CAt extract, which showed marked inhibitory effects on HCC cells in a dose-dependent manner; in contrast, the CAt extract treatment was less cytotoxic to normal cells. In addition, our results indicate that the CAt extract induced apoptosis via caspase-dependent and independent apoptosis pathways. Furthermore, the CAt extract inhibited HCC tumor cell growth by restraining cell cycle progression, and it reduced the signaling of the AKT, ERK1/2, and p38 pathways. In the xenograft model, the CAt extract suppressed HCC tumor cell growth and prolonged lifespan by inhibiting PCNA protein expression, repressing part of the VEGF-induced autocrine pathway, and triggering strong expression of cleaved caspase-3, which contributed to cell apoptosis. Moreover, the CAt extract did not induce any obvious changes in pathological morphology or body weight, suggesting it had no toxicity. CAt extract exerted anti-tumor effects on HCC in vitro and in vivo. Thus, CAt extract could be used as a potential anti-cancer therapeutic agent against HCC.

Extract of Pogostemon cablin Possesses Potent Anticancer Activity against Colorectal Cancer Cells In Vitro and In Vivo.

Pogostemon cablin (PCa), an herb used in traditional Chinese medicine, is routinely used in the amelioration of different types of gastrointestinal discomfort. However, the mechanisms underlying the cancer suppression activity of PCa in colorectal cancer (CRC) cells have yet to be clarified. The aim of this study was to investigate the anticancer effects of PCa, specifically the induction of apoptosis in CRC cells. The growth inhibition curve of CRC cells following exposure to PCa was detected by an MTT assay. Moreover, PCa combined with 5-FU revealed a synergic effect of decreased cell viability. PCa inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase and cell apoptosis through regulation of associated protein expression. An in vivo study showed that PCa suppressed the growth of CRC via induction of cell apoptosis with no significant change in body weight or organ histology. Our results demonstrated that PCa inhibits the growth of CRC cells and induces apoptosis in vitro and in vivo, which suggests the potential applicability of PCa as an anticancer agent.

Suppression of oral cancer by induction of cell cycle arrest and apoptosis using Juniperus communis extract.

The oral cancer incidence rate is slowly increasing and is now the fifth leading cause of cancer-related death due to its high metastasis and recurrence rate. Juniperus communis is used as a traditional Chinese medicine and has been proven to have anti-cancer activity against neuroblastomas. In the present study, we further investigated the anti-cancer mechanisms of J. communis extract (JCo) on oral cancer and evaluated the synergistic effects of JCo combined with 5-fluorouracil (5-FU). We found that JCo inhibited oral cancer cell growth, and that JCo might be less cytotoxic to normal cells than to cancer cells. After JCo treatment, cell cycle arrest was observed at the G0/G1 phase through modulation of p53/p21 and Rb signaling. JCo also caused an increase in the sub-G1 phase and cell apoptosis via the intrinsic and extrinsic apoptosis pathways. JCo combined with 5-FU presented a synergistic effect to reduce cell viability. In conclusion, JCo inhibited oral cancer cell growth by inducing cell cycle arrest and activating cell apoptosis, and JCo significantly synergized with 5-FU. JCo might have the potential to be an adjuvant and a new therapeutic drug for oral cancer treatment.

Extract of Juniperus indica Bertol Synergizes with Cisplatin to Inhibit Oral Cancer Cell Growth via Repression of Cell Cycle Progression and Activation of the Caspase Cascade.

Oral cancer-a type of head and neck cancer-is estimated to be the fifth most common cancer in Taiwan. However, efficacious therapies for oral cancer are still lacking due to drug resistance and recurrence. Consequently, the identification of new anticancer agents for clinical treatment is needed. Juniperus indica Bertol is a plant of the Juniperus genus often used as a treatment in traditional medicine due to its anti-inflammatory, antibacterial and diuretic functions. The biofunctions of Juniperus indica Bertol including its anticancer potential, have not been fully explored. As a result, the aim of this research was to investigate the anticancer activity of Juniperus indica Bertol extract (JIB extract) and determine whether JIB extract has synergistic effects with cisplatin in oral cancer. These results are the first to demonstrate that JIB extract exhibits anticancer capacity and synergizes with cisplatin to treat oral cancer. Our findings indicate that JIB extract has a potential to develop anticancer agent and chemo therapeutic adjuvant for oral cancer.

YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance by suppressing caspase-mediated apoptosis.

The objective of this study was to characterize the oncogenic actions of a recently identified cancer-associated gene YWHAZ (also named as 14-3-3 ζ/δ) in urothelial carcinomas of the urinary bladder (UCUB). A genome-wide study revealed YWHAZ to be involved in the amplicon at 8q22.3, and its genetic amplification was detected predominantly in muscle-invasive bladder cancer (MIBC). Immunohistochemical staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis and STRING network studies suggested involvement of YWHAZ in regulating caspase-mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low endogenous YWHAZ levels boosted cell resistance to doxorubicin and cisplatin, as well as to ionizing radiation. Conversely, YWHAZ-knockdown using specific shRNA in cells with high endogenous YWHAZ levels diminished survival activity, suppressing cell growth and increasing cell death. Our findings confirm the essential role played by YWHAZ in sustaining cell proliferation during chemo/radiotherapy. Treatments based on anti-YWHAZ strategies may thus be beneficial for UCUB patients overexpressing YWHAZ. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

DNA methylation-regulated microRNA pathways in ovarian serous cystadenocarcinoma: A meta-analysis.

Epigenetic regulation has been linked to the initiation and progression of cancer. Aberrant expression of microRNAs (miRNAs) is one such mechanism that can activate or silence oncogenes (OCGs) and tumor suppressor genes (TSGs) in cells. A growing number of studies suggest that miRNA expression can be regulated by methylation modification, thus triggering cancer development. However, there is no comprehensive in silico study concerning miRNA regulation by direct DNA methylation in cancer. Ovarian serous cystadenocarcinoma (OSC) was therefore chosen as a tumor model for the present work. Twelve batches of OSC data, with at least 35 patient samples in each batch, were obtained from The Cancer Genome Atlas (TCGA) database. The Spearman rank correlation coefficient (SRCC) was used to quantify the correlation between the CpG DNA methylation level and miRNA expression level. Meta-analysis was performed to reduce the effects of biological heterogeneity among different batches. MiRNA-target interactions were also inferred by computing SRCC and meta-analysis to assess the correlation between miRNA expression and cancer-associated gene expression and the interactions were further validated by a query against the miRTarBase database. A total of 26 potential epigenetic-regulated miRNA genes that can target OCGs or TSGs in OSC were found to show biological relevance between DNA methylation and miRNA gene expression. Furthermore, some of the identified DNA-methylated miRNA genes; for instance, the miR-200 family, were previously identified as epigenetic-regulated miRNAs and correlated with poor survival of ovarian cancer. We also found that several miRNA target genes, BTG3, NDN, HTRA3, CDC25A, and HMGA2 were also related to the poor outcomes in ovarian cancer. The present study proposed a systematic strategy to construct highly confident epigenetic-regulated miRNA pathways for OSC. The findings are validated and are in line with the literature. The inclusion of direct DNA methylated miRNA events may offer another layer of explanation that along with genetics can give a better understanding of the carcinogenesis process.

Genetic variations of MUC17 are associated with endometriosis development and related infertility.

BACKGROUND: Genetic alterations of mucin genes, such as MUC2 and MUC4, were previously identified to be associated with endometriosis and related infertility. Additionally, gene expression profiling has confirmed MUC17 to be overexpressed in mucinous ovarian carcinoma; however, its associated risk for endometriosis remains unclear. This study was focused on the potential impact of genetic variations in MUC17 on endometriosis development and associated clinical features. METHODS: The study subjects included 189 female Taiwanese patients with pathology-proven endometriosis and 191 healthy Taiwanese women as controls. Five single-nucleotide polymorphisms (rs4729645, rs10953316, rs74974199, rs4729655, and rs4729656) within the MUC17 gene were selected and genotyped using the Taqman genotyping assay to examine the allele frequency and genotype distributions of MUC17 polymorphisms. RESULTS: Genotyping revealed that the A allele at rs10953316 in MUC17 was a protective genetic factor in endometriosis development (p = 0.008; OR = 0.53; 95% CI: 0.36-0.79). Genetic variation of rs4729655 protected patients against endometriosis-induced infertility, but was associated with a higher cancer antigen 125 (CA125) level. Base-pairing analysis, called MaxExpect, predicted an additional loop in the mRNA structure caused by rs10953316 polymorphism, possibly influencing ribosome sliding and translation efficiency. Such predictions were confirmed by immunohistochemistry that patients with AA genotype at rs10953316 showed low MUC17 levels in their endometrium, patients with GA genotype showed moderate levels, and strong staining could be found in patients with GG genotype. CONCLUSIONS: MUC17 polymorphisms are involved in endometriosis development and the associated infertility in the Taiwanese population.

BMPR1B up-regulation via a miRNA binding site variation defines endometriosis susceptibility and CA125 levels.

BACKGROUND: Bone morphogenetic protein receptor I B (BMPR1B) is a transmembrane receptor mediating TGF-ß signal transduction. Recent studies indicate a tumor suppressor role for BMPR1B in ovarian cancer. Polymorphism at BMPR1B 3'UTR within the miR-125b binding site alters its binding affinity toward the miRNA, which may result in insufficient post-transcriptional repression. METHODS: Single-nucleotide polymorphisms rs1970801, rs1434536, and rs11097457 near the miR-125b binding site in BMPR1B were genotyped by Taqman assay on 193 endometriosis patients and 202 healthy controls. BMPR1B and CA125 levels in ectopic endometrial tissues were evaluated by quantitative PCR and immunohistochemistry. Luciferase reporter assay was utilized to verify regulatory roles of BMPR1B 3'UTR with allelic variants of rs1434536 in a cell line model. Cell proliferation and migration were recorded, while expression of BMPR1B, CA125, glucocorticoid receptor (GCCR) and IL-1ß were measured by quantitative PCR in endometrial cells transfected with wild-type or mutated miR-125b. RESULTS: This study found two endometriosis-associated SNPs, rs1434536 (P = 0.010) and rs1970801 (P = 0.0087), located within and next to a miR-125b binding site on BMPR1B. Interestingly, patients with homozygous variant alleles at rs1434536 showed significantly lower serum CA125 levels. Immunohistochemistry staining further confirmed inverse correlation between BMPR1B and CA125 levels in three rs1434536 genotypes. Cell assays demonstrated the variant allele of rs1434536 up-regulating BMPR1B at both mRNA and protein levels, which negatively correlated with CA125 and IL-1ß levels. Disruption of the binding between miR-125b and BMPR1B hampered abnormal cell proliferation. CONCLUSIONS: SNPs of BMPR1B within and next to the miR-125b binding site manifested strong correlation with endometriosis development in a Taiwanese cohort. Disrupting the binding of miR-125b toward BMPR1B would increase protein expression, diminishing abnormal cell proliferation as well as serum and cellular CA125 levels. Genetic variation at the miR-125b binding site may play functional roles to protect against endometriosis progression.

Analysis of the NCI-60 dataset for cancer-related microRNA and mRNA using expression profiles.

BACKGROUND: Recent studies have indicated that microRNA (miRNA) may play an oncogenic or tumor suppressor role in human cancer. To study the regulatory role of miRNAs in tumorigenesis, an integrated platform has been set up to provide a user friendly interface for query. The main advantage of the present platform is that all the miRNA target genes' information and disease records are drawn from experimentally verified or high confidence records. RESULTS: MiRNA target gene results are annotated with reference to the disease gene as well as the pathway database. The correlation strength between miRNA and target gene expression profile is quantified by computing the correlation coefficient using the NCI-60 expression profiling data. Comprehensive analysis of the NCI-60 data found that the cumulative percentage of negative correlation coefficients for cleavage regulation is slightly higher than its positive counterpart; which indicated that the mRNA degradation mechanism is slightly dominant. In addition, the RNAHybrid and TargetScans scores are computed which potentially served as quantitative estimators for miRNA-mRNA binding events. Three scores are defined for each miRNA-mRNA pair, which are based on the disease gene and pathway information. These three scores allow user to sort out high confidence cancer-related miRNA-mRNA pairs. Statistical tests were applied to investigate the relations of three chromosomal features, i.e., CpG island, fragile site, and miRNA cluster, with cancer-related miRNAs. A web-based interface has been set up for query, which can be accessed at: http://ppi.bioinfo.asia.edu.tw/mirna_target/ CONCLUSIONS: The main advantage of the present platform on miRNA-mRNA targeting information is that all the target genes' information and disease records are experimentally verified. Although this may limit the number of miRNA-mRNA relationships, the results provided here are more solid and have fewer false positive events. Certain novel cancer-related miRNA-mRNA pairs are identified and confirmed in the literature. Fisher's exact test suggests that CpG island and fragile site associated miRNAs tend to associate with cancer formation. In summary, the present platform provides an easy means of investigating cancer-related miRNAs.

Electroporation-mediated IL-12 gene therapy in a transplantable canine cancer model.

Interleukin-12 (IL-12) is effective in treating many types of rodent tumors, but has been unsuccessful in most human clinical trials, suggesting that animal models of more clinical relevance are required for evaluating human cancer immunotherapy. Herein, we report on the effectiveness of gene therapy with plasmid encoding human IL-12 (pIL-12) through in vivo electroporation in the treatment of beagles with a canine tumor, the canine transmissible venereal tumor (CTVT). The optimal electroporation conditions for gene transfer into CTVTs were tested by luciferase activity and determined to be a voltage of 200 V and duration of 50 msec, with the number of shocks set at 10 pulses, and the use of an electrode with 2 needles. Under these conditions, intratumoral administration of as little as 0.1 mg pIL-12 followed by electroporation significantly inhibited the growth of well-established tumors and eventually led to complete tumor regression. Furthermore, local pIL-12 treatment also induced a strong systemic effect that prevented new tumor growth and cured established tumors at distant locations. Intratumoral administration of pIL-12 greatly elevated the IL-12 level in the tumor masses, but produced only a trace amount in the serum. A high level of IFN-gamma mRNA was also detected in the treated tumor masses. pIL-12 gene therapy attracted significantly more lymphocytes infiltrating the tumors, including CD4(+) and CD8(+) T cells, and the surface expression of MHC I and MHC II molecules on CTVT cells was greatly increased after pIL-12 therapy. This treatment also induced apoptosis of the tumor cells as detected by Annexin V. More importantly, delivery of pIL-12 with intratumoral electroporation did not result in any detectable toxicity in the dogs. We conclude that intratumoral electroporation of the pIL-12 gene could cause profound immunologic host responses and efficiently treat CTVT in beagle dogs. The results also indicate that CTVT is an excellent large animal cancer model for testing immunogene therapies mediated by electroporation.