Expression Levels Of ABCG2 Research Articles

Abstract 5146: ATP-binding cassette transporter, ABCG2, activation is responsible for chemo-resistant, SIK3 low expressed ovarian cancer

Abstract Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among the gynecological cancers because of chemo-resistant development. Materials and Method: The human EOC cell lines, SKOV3, OVRCA4, were obtained from the American Type Culture Collection (ATCC). For determine ATP-binding cassette transporters and SIK3 expression, Western blot and Quantitative real-time polymerase chain reaction (qRT-PCR) were used. We used a lentiviral small-hairpin RNA (shRNA) system to knock down SIK3 in the ovarian cancer cell lines. Microarray data derived from SKOV3, OVRCA4 cells expressing sh-luc, sh-SIK3 (01), and sh-SIK3 (63) were analyzed by MetaCore and Ingenuity Pathways Analysis (IPA). Top 10 up-and down-regulated genes and other metastasis-related genes and 5 most highly enriched networks were identified. For determining cell viability when cells treated with cisplatin and Taxol, WST-1 assay was used. Immunohistochemistry and quantification of ABCG2 and SIK3 were performed in EOC tissues. Statistical analysis of OS and PFS were performed using SPSS statistical software (Version 22.0; IBM Corp, Armonk, NY). Survival curves were generated using the Kaplan-Meier method, and differences in survival were assessed using the log-rank test. Results: Knockdown SIK3 expression upregulates ABCG2 expression, activity as well as resistance to chemotherapeutic agents. The ABCG2 and SIK3 expression levels in 204 EOC patient were mutually reversed. In all stages of the disease, high expression of SIK3 (SIK3-H) was associated with a significant better OS, PFS, compared with low expression of SIK3 (SIK3-L) (127.0 vs 46.0 months, 66.0 months vs 26.0 months respectively). Patients with SIK3-L and ABCG2-H carried the worst prognosis. Conclusion: (1) High expression of SIK3 indicates a better prognosis in primary ovarian cancer and serous type disease, especially in advanced serous disease. (2) Ovarian cancer patients with SIK3-L and ABCG2-H expression carried the worst prognosis due to chemoresistance Citation Format: Keng-Fu Hsu, Yu-Ling Liang, Chin-Han Wu, Neng-Yao Shih. ATP-binding cassette transporter, ABCG2, activation is responsible for chemo-resistant, SIK3 low expressed ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5146.

The effect of hericium polysaccharides combined with aspirin on the expression of CD133 and ABCG2 in lung cancer

Background: The study was designed to investigate the effects of aspirin and hericium polysaccharides (HP) on the expression of CD133 and ABCG2 in lung cancer growth. Methods: In vitro , A549 cells were treated with aspirin alone, HP alone, and a combination of both substances. Cell proliferation and the expression of CD133 and ABCG2 were examined using the CCK-8 assay, colony formation assay, and western blot. In vivo , eight C57BL/6J male mice were randomly selected as a blank group. The remaining 42 were inoculated with A549 cells to establish tumor-bearing model. The tumor-bearing mice were giving aspirin alone, HP alone, and a combination of both substances and sacrificed after 30 days of drug treatment. After tumor volumes were calculated, and tumor tissues were weighed, Western blot and quantitative fluorescence PCR were used to detect the expression of CD133 and ABCG2 in tumor tissues from different groups. Results: Aspirin alone, HP alone and combined aspirin and HP inhibited cell proliferation significantly. Western blot results showed that Aspirin alone, HP alone and combined HP and aspirin inhibited the expression levels of CD133 significantly, but HP alone did not inhibit the expression levels of ABCG2. Combined HP and aspirin are synergistic. In vivo , HP alone, aspirin alone, and combined aspirin and HP inhibited tumor volume and tumor weights significantly. According to results from the Western blot and PCR analyses, HP alone, aspirin alone, and combined HP and aspirin inhibited the expression levels of CD133 significantly, but HP alone did not inhibit the expression levels of ABCG2. Combined aspirin and HP applications are synergistic. Conclusions: The combined effect of aspirin and HP inhibited tumor growth significantly by obstructing CD133 and ABCG2 expression in lung cancer.

Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance

Ko143, a potent ABCG2 inhibitor that reverses multidrug resistance in cancer, cannot be used clinically due to its unsuitable metabolic stability. We identified benzoyl indoles as reversal agents that reversed ABCG2-mediated multidrug resistance (MDR), with synthetic tractability and enhanced metabolic stability compared to Ko143. Bisbenzoyl indole 2 and monobenzoyl indole 8 significantly increased the accumulation of mitoxantrone (MX) in ABCG2-overexpressing NCI–H460/MX20 cells, and sensitized NCI–H460/MX20 cells to mitoxantrone. Mechanistic studies were conducted by [3H]-MX accumulation assay, Western blot analysis, immunofluorescence analysis and ABCG2 ATPase assay. The results revealed that the reversal efficacies of compounds 2 and 8 were not due to an alteration in the expression level or localization of ABCG2 in ABCG2-overexpressing cell lines. Instead, compounds 2 and 8 significantly stimulated the ATP hydrolysis of ABCG2 transporter, suggesting that these compounds could be competitive substrates of ABCG2 transporter. Overall, the results of our study indicated that compounds 2 and 8 significantly reversed ABCG2-mediated MDR by blocking the efflux of anticancer drugs.

Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

The effect of hericium polysaccharides combined with aspirin on the expression of CD133 and ABCG2 in lung cancer.

BackgroundThe study was designed to investigate the effects of aspirin and hericium polysaccharides (HP) on the expression of CD133 and ABCG2 in lung cancer growth.MethodsIn vitro, A549 cells were treated with aspirin alone, HP alone, and a combination of both substances. Cell proliferation and the expression of CD133 and ABCG2 were examined using the CCK-8 assay, colony formation assay, and western blot. In vivo, eight C57BL/6J male mice were randomly selected as a blank group. The remaining 42 were inoculated with A549 cells to establish tumor-bearing model. The tumor-bearing mice were giving aspirin alone, HP alone, and a combination of both substances and sacrificed after 30 days of drug treatment. After tumor volumes were calculated, and tumor tissues were weighed, Western blot and quantitative fluorescence PCR were used to detect the expression of CD133 and ABCG2 in tumor tissues from different groups.ResultsAspirin alone, HP alone and combined aspirin and HP inhibited cell proliferation significantly. Western blot results showed that Aspirin alone, HP alone and combined HP and aspirin inhibited the expression levels of CD133 significantly, but HP alone did not inhibit the expression levels of ABCG2. Combined HP and aspirin are synergistic. In vivo, HP alone, aspirin alone, and combined aspirin and HP inhibited tumor volume and tumor weights significantly. According to results from the Western blot and PCR analyses, HP alone, aspirin alone, and combined HP and aspirin inhibited the expression levels of CD133 significantly, but HP alone did not inhibit the expression levels of ABCG2. Combined aspirin and HP applications are synergistic.ConclusionsThe combined effect of aspirin and HP inhibited tumor growth significantly by obstructing CD133 and ABCG2 expression in lung cancer.

Downregulated Salt-inducible Kinase 3 Expression Promotes Chemoresistance in Serous Ovarian Cancer via the ATP-binding Cassette Protein ABCG2.

Background: Epithelial ovarian cancer (EOC) has a high tumor-associated mortality rate among gynecological cancers. Although CA125 is a well-studied biomarker for ovarian cancer, it is also elevated under numerous conditions, resulting in decreased specificity. Recently, we identified a novel tumor-associated antigen, salt-inducible kinase 3 (SIK3), during tumorigenesis in ovarian cancer. However, the association between SIK3 expression and patient outcomes in ovarian cancer remains unclear.Materials and Methods: We collected EOC samples from 204 patients and examined tumor SIK3 expression by immunohistochemistry (IHC) and CA125 expression in tumors and serum. The expression levels of SIK3 and CA125 were correlated with patient survival. SIK3 expression was silenced with SIK3-specific shRNAs to investigate the possible mechanisms related to chemoresistance in serous-type ovarian cancer cell lines OVCAR4 and SKOV3.Results: In advanced-stage serous ovarian cancer, patients with low SIK3 expression have poorer overall survival (OS) and progression-free survival (PFS) than patients with high SIK3 expression. Ovarian cancer cells with SIK3 knockdown display increased chemoresistance to Taxol plus cisplatin treatment, which is associated with the upregulation of the ABCG2 transporter. In addition, in serous ovarian cancer, SIK3 expression is inversely correlated to ABCG2 expression, and patients with low SIK3 and high ABCG2 expression have worse prognosis than patients with high SIK3 and low ABCG2 expression.Conclusion: Our results demonstrated that serous EOC patients with low SIK3 expression have poor prognosis, which is associated with chemoresistance mediated by ABCG2 upregulation. SIK3 and ABCG2 expression levels may be potential prognostic markers to predict the outcome in serous EOC patients.

ABCA7 Downregulation Modifies Cellular Cholesterol Homeostasis and Decreases Amyloid-β Peptide Efflux in an in vitro Model of the Blood-Brain Barrier.

The role of ABCA7 in brain homeostasis and Alzheimer's disease (AD) is currently under intense scrutiny, since it has been reported that polymorphisms in the Abca7 gene and a loss of function of the protein are closely linked to excessive accumulation of amyloid peptides and disturbed cholesterol homeostasis. The blood-brain barrier (BBB), which isolates the brain from the blood compartment, is involved in both of these processes. We therefore hypothesized that ABCA7 downregulation might affect cholesterol and amyloid exchanges at the BBB. Using siRNA and primary cultures of mouse endothelial cells purified from brain microvessels and seeded on Transwell ® inserts, we investigated the role of ABCA7 in cholesterol and amyloid exchanges across the BBB. Our results showed that a decrease in ABCA7 expression at the BBB provokes in vitro a reduction in ABCA1 expression and a decrease in APOE secretion. In vitro, these decreases reduce cholesterol exchange across the BBB, particularly for high-density lipoproteins and ApoA-I particles. When ABCA7 was absent, we observed a reduction in Aβ peptide basolateral-to-apical transport in the presence of ApoA-I, with non-significant changes in the expression levels of Rage, Lrp1, Abcb1, Abcc1, and Abcg2. Our study in murine BBB model highlighted a putative new role for ABCA7 in AD via the protein's involvement in cholesterol metabolism and amyloid clearance at the BBB.

Transcriptome analysis of ABCB1, ABCG2 and the BCL2/BAX ratio in refractory and relapsed canine lymphomas under treatment and rescue protocol

Abstract The main problems that cause unresponsiveness to an anti-neoplastic drug are the overexpression of drug resistant and anti-apoptotic proteins in tumor cells. In a rescue protocol we evaluated the ability of toceranib phosphate concurrent with lomustine (CCNU) or L-asparaginase and vincristine to decrease drug resistant and apoptotic proteins in relapsed and refractory canine lymphomas. The peripheral blood samples were collected before and after the rescue treatment from fourteen dogs that were refractory to cyclophosphamide-vincristine-prednisolone (COP) or COP-doxorubicin (CHOP) treatment and had recurrent multicentric lymphoma. The mRNA expression level of ABCB1, ABCG2, Bcl2 and Bax were determined by quantitative real-time PCR. The fold-change in ABCB1, ABCG2, Bcl2 and Bax mRNA levels were analyzed in correlation with the progression-free survival (PFS). After the rescue treatment, the ABCB1 and ABCG2 mRNA expression levels were 1.57- and 1.85-fold lower (p = 0.4 and p = 0.87), respectively, compared to pre-treatment. Bcl2/Bax ratio was numerically but not significantly decreased 1.02-fold (p = 0.74). The overall response rate of this protocol was 50% with a median PFS of 79 days (range 14-207 days). The low medians of relative expression levels of ABCB1, ABCG2 and Bcl2/Bax ratio group did not correlate with the clinical outcomes when compared to the high medians of relative expression levels, and likewise with the clinical stage, immunophenotype, histological grade and sub-stage. Therefore, the administration of a rescue drug with toceranib phosphate might be beneficial in refractory and relapsed canine lymphoma.

Co-expression of ATP binding cassette transporters is associated with poor prognosis in acute myeloid leukemia

Chemotherapy failure remains a challenge when treating patients with acute myeloid leukemia (AML), who often suffer from persistent or relapsed disease. The multidrug resistance (MDR) mediated by efflux transporters of the ATP binding cassette (ABC) superfamily is a major obstacle for successful chemotherapy. The present study aimed to elucidate whether the expression of ABC transporters was associated with prognostic factors and responses to chemotherapy in patients with AML, with particular focus on whether co-expression of multiple ABC transporters resulted in a worse prognosis. In the present study, the mRNA expression levels of ABC transporters ABCB1, ABCB4, ABCC1, ABCC4 and ABCG2 in bone marrow (BM) mononuclear cell (MNC) samples from 96 de novo patients with AML and in the peripheral blood (PB) MNC samples from 22 normal individuals were investigated using reverse transcription-quantitative polymerase chain reaction analysis. It was revealed that ABCB1, ABCC1, ABCC4 and ABCG2 were expressed at higher levels in patients with AML compared with normal individuals, whereas ABCB4 had a lower expression level. The expression of ABCB4 in patients with AML was significantly lower than in normal individuals (P<0.001). Patients risk status was associated with ABCB1 (P=0.037), ABCC1 (P=0.047), ABCC4 (P=0.015) and ABCG2 (P=0.027). The 4 genes were expressed a significantly higher levels in the poor response group compared with the good response group (ABCB1, P=0.014; ABCC1, P=0.021; ABCC4, P=0.005; ABCG2, P=0.009). The overexpression of the 4 ABC transporters and the complete remission rate were inversely correlated (P<0.001). These results suggest that the co-expression of multiple ABC transporters may contribute to a worse prognosis in AML.

Dacomitinib potentiates the efficacy of conventional chemotherapeutic agents via inhibiting the drug efflux function of ABCG2 in vitro and in vivo

BackgroundATP-binding cassette subfamily G member 2 (ABCG2), a member of the ABC transporter superfamily proteins, mediates multidrug resistance (MDR) by transporting substrate anticancer drugs out of cancer cells and decreasing their intracellular accumulation. MDR is a major hurdle to successful chemotherapy. A logical approach to overcome MDR is to inhibit the transporter. However, no safe and effective MDR inhibitor has been approved in the clinic.MethodsThe MTT assay was used to evaluate cell cytotoxicity and MDR reversal effect. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The H460/MX20 cell xenograft model was established to evaluate the enhancement of anticancer efficacy of topotecan by dacomitinib in vivo. To ascertain the interaction of dacomitinib with the substrate binding sites of ABCG2, the competition of dacomitinib for photolabeling of ABCG2 with [125I]- iodoarylazidoprazosin (IAAP) was performed. Vanadate-sensitive ATPase activity of ABCG2 was measured in the presence of a range of different concentrations of dacomitinib to evaluate the effect of dacomitinib on ATP hydrolysis as the energy source of the transporter. A flow cytometry-based assay and western blotting were employed to study whether dacomitininb could inhibit the expression level of ABCG2. The mRNA expression levels of ABCG2 were analyzed by real-time quantitative RT-PCR. The protein expression level of AKT, ERK and their phosphorylations were detected by Western blotting.ResultsHere, we found that dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor (TKI) in phase III clinical trial, could enhance the efficacy of conventional chemotherapeutic agents specifically in ABCG2-overexpressing MDR cancer cells but not in the parental sensitive cells. Dacomitinib was found to significantly increase the accumulation of ABCG2 probe substrates [doxorubicin (DOX),Rhodamine 123 (Rho 123) and Hoechst 33342] by inhibiting the transporter efflux function. Moreover, dacomitinib stimulated ABCG2 ATPase activity and competed with [125I]-IAAP photolabeling of ABCG2 in a concentration-dependent manner. However, dacomitinib did not alter ABCG2 expression at protein and mRNA levels or inhibit ErbB downstream signaling of AKT and ERK. Importantly, dacomitinib significantly enhanced the efficacy of topotecan in ABCG2-overexpressing H460/MX20 cell xenografts in nude mice without incurring additional toxicity.ConclusionsThese results suggest that dacomitinib reverses ABCG2-mediated MDR by inhibiting ABCG2 efflux function and increasing intracellular accumulation of anticancer agents. Our findings advocate further clinical investigation of combinations of dacomitinib and conventional chemotherapy in cancer patients with ABCG2-overexpressing MDR tumors.

Enhancement of the Effect of Methyl Pyropheophorbide-a-Mediated Photodynamic Therapy was Achieved by Increasing ROS through Inhibition of Nrf2-HO-1 or Nrf2-ABCG2 Signaling.

Emerging evidence indicates that the transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays an essential role in cellular defense against oxidative stress; its activation has been related to cytoprotection. Here, we investigated the role of Nrf2 in improving the efficacy of methyl pyropheophorbide-amediated photodynamic therapy (Mppa-PDT) via the downregulation of Nrf2. Human ovarian cancer A2780 cells and SKOV3 cells were treated with Mppa-PDT and siRNA transfection was performed to inhibit Nrf2. After treated with siRNA and Mppa-PDT, the cell viability was examined with CCK-8 assay; cell apoptosis was detected tested by flow cytometry with Annexin V-FITC/PI; the celluar reactive oxygen species (ROS) and mitochondrial membrane potential were measured with DCFHDA and JC-1 staining; expression of protein was assessed by western blot analysis. We found that Nrf2 translocated from the cytoplasm to the nucleus in vitro and in vivo, and the expression of Nrf2 and P-Nrf2 increased through a possible mechanism regulated by mitogen-activated protein kinase (MAPK) after Mppa-PDT treatment. Furthermore, cytotoxicity and apoptosis induced by Mppa-PDT increased after Nrf2down-regulation. Nrf2 down -regulation increased reactive oxygen species (ROS) levels by attenuating antioxidants or pumping Mppa out of cells,which resulted from the inhibition of Nrf2-HO-1 or Nrf2- ABCG2 signaling. In addition, SKOV3 cells exhibited increased resistance to Mppa-PDT, and the expression levels of P-Nrf2 and ABCG2 were higher in SKOV3 cells than in A2780 cells, suggesting that Nrf2-ABCG2 signaling might be involved in the intrinsic resistanceto Mppa-PDT. These results provided evidence that Nrf2 down-regulation can enhance the effect of Mppa-PDT.

Clinically relevant interactions of anti-apoptotic Bcl-2 protein inhibitors with ABC transporters.

Antiproliferative effects of ABT-263 and ABT-199 might be reduced by ABCB1 or ABCG2, however, this effect depends on transporter expression levels. Since the expression levels of ABCB1 and ABCG2 are rarely high in clinical samples, their contribution to the overall resistance to ABT-263 or ABT-199 is probably low. Inhibition study revealed that ABT-199, but not ABT-263, fully inhibited low expression level of ABCG2. Our data suggest that ABT-199 should be evaluated beyond its original application as an inhibitor of the ABCG2 transporter in clinical settings.

MicroRNA-655-3p regulates Echinacea purpurea mediated activation of ABCG2

1. The aim of this study was to investigate the regulatory effect of Echinacea purpurea (EP) on efflux transporters ABCB1 and ABCG2 and to identify specific microRNAs contributing to their post-transcriptional regulation.2. ABCB1 and ABCG2 levels were assessed in human hepatoblastoma HepG2 cells treated with 50 µg/mL methanolic extract of commercial EP capsules for different durations. The microRNA expression profile of HepG2 cells after EP treatment was evaluated and in silico target prediction was subsequently conducted to identify specific microRNAs with binding sites in the 3′-UTR of ABCB1 and ABCG2. Luciferase reporter gene assays and site-directed mutagenesis were used to confirm the binding site of identified microRNA within the 3′-UTR of the target gene.3. EP increased ABCB1 (10-fold ± 3.4, p < 0.001) and ABCG2 (2.7-fold ± 0.5, p < 0.01) mRNA levels after 12 h exposure. Twenty-four microRNAs showed significant expression differences at all durations of exposure to EP. MiR-655-3p showed a 6.79-fold decrease in expression after 12 h exposure compared to 0 h, was predicted in silico to bind ABCG2 3′-UTR and showed a significant negative correlation (p = 0.01) to ABCG2 expression level. The binding of miR-655-3p to ABCG2 3′-UTR was confirmed by reporter gene assays (reduction of reporter gene activity to 60%; p = 0.0001).4. These results suggest that EP regulates ABCG2 expression via downregulation of miR-655-3p in the liver cells. Thus, miR-655-3p downregulation could be applied to predict EP mediated drug interactions.

Effects of aqueous extracts of gecko on stemness of hepatocellular carcinoma

To explore the effects and mechanism of aqueous extracts of gecko on cancer stem cells properties of hepatocellular carcinoma. In vitro, MTT assay was used to detect the cells growth in Huh7 and Hep3B. Spheroid-forming assay and flow cytometry were performed to observe the the stemness of Huh7 and Hep3B cells. The protein expressions of β-catenin, CD44, c-Myc, CCND1, Sox2, Oct4, Nanog and ABCG2 were detected by Western blot. Interacting proteins were detected by co-immunoprecipitation; and a subcutaneous xenograft model was used to detect the stemness of hepatoma carcinoma cells. The results indicated that aqueous extracts of gecko induced cell growth inhibition in a dose- and time-dependent manner, with the IC₅₀ of (0.750±0.112) g•mL⁻¹ for Huh7 and (0.454±0.039) g•mL⁻¹ for Hep3B, respectively. The number and size of tumor spheres formed by hepatoma carcinoma cells were decreased after treatment by aqueous extracts of gecko(P<0.05); the proportions of cells staining with putative markers for cancer stem cells, such as CD133 and CD44, were decreased(P<0.05). After treatment with aqueous extracts of gecko, the expression levels of β-catenin, CD44, c-Myc, CCND1, Sox2, Oct4, Nanog and ABCG2 were decreased. Co-immunoprecipitation results showed that the aqueous extracts of gecko could inhibit the interaction between LRP6 and Frizzled6, indicating that the aqueous extracts of gecko could inhibit the proliferation of hepatoma cells, the formation of tumor spheres and the proportion of tumor stem cells, and inhibit the Wnt signaling pathway by targeting LRP6 to prevent the formation of LRP6 and Frizzled6 complexes.

HM015k, a Novel Silybin Derivative, Multi-Targets Metastatic Ovarian Cancer Cells and Is Safe in Zebrafish Toxicity Studies.

This study was designed to determine the in vitro mechanisms by which the novel silybin derivative, (E)-3-(3-(benzyloxy) phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (HM015k or 15k), produces its anticancer efficacy in ovarian cancer cells. Compound 15k induced apoptosis in ovarian cancer cells in a time-dependent manner by significantly upregulating the expression of Bax and Bak and downregulating the expression of Bcl-2. Interestingly, 15k induced the cleavage of Bax p21 into its more efficacious cleaved form, Bax p18. In addition, caspase 3 and caspase 9 were cleaved to their active forms, inducing the cleavage of poly ADP ribose polymerase (PARP) and β-catenin. Furthermore, in OV2008 cells, 15k induced significant cleavage in nuclear β-catenin to primarily inactive fragments of lower molecular weight. Furthermore, 15k reversed the metastatic potential of OV2008 cells by inhibiting their migration and invasiveness. The mesenchymal phenotype in OV2008 was reversed by 15k, causing cells to be rounder with epithelial—like phenotypes. The 15k-induced reversal was further confirmed by significant upregulation of the E-cadherin expression, an epithelial marker, while N-cadherin, a mesenchymal marker, was downregulated in OV2008 cells. Compound 15k inhibited the expression of the oncogenic c-Myc protein, downregulated proteins DVL3 and DVL2 and significantly upregulated cyclin B1. Also, 15k significantly downregulated the expression levels of ABCG2 and ABCB1 transporters in resistant ABCG2 overexpressing H460/MX20 and resistant ABCB1 overexpressing MDCK/MDR1 cells, respectively. Finally, 15k was safe in zebrafish in vivo model at concentrations up to 10 μM and induced no major toxicities in cardiac, morphology and swimming position parameters. Overall, 15k is a multi-targeted inhibitor with efficacy against metastatic and resistant ovarian cancer. Future in vivo studies will be conducted to determine the efficacy of 15k in tumor-bearing animals.

Non-small cell lung cancer PC-9 cells exhibit increased sensitivity to gemcitabine and vinorelbine upon acquiring resistance to EGFR-tyrosine kinase inhibitors.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) are widely used for the treatment of non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations. However, lung cancer cells inevitably acquire resistance to these EGFR-TKIs. The majority of patients whose lung cancer acquires resistance to EGFR-TKIs are subjected to treatment using cytotoxic agents. The present study aimed to determine if lung cancer cells acquiring resistance to EGFR-TKIs also develop altered sensitivity to cytotoxic agents. It was revealed that lung cancer cells that had developed resistance to EGFR-TKIs had increased sensitivity to gemcitabine and vinorelbine compared with EGFR-TKI naïve cells. The expression levels of ATP-binding cassette (ABC) transporter genes, including ABCC3, ABCC5 and ABCG2, were observed to be commonly repressed in EGFR-TKI-resistant cells. In addition, two cases were identified in which gemcitabine and vinorelbine exerted marked responses to lung cancers that had acquired resistance to EGFR-TKIs, even with late-line treatment. Therefore, it was proposed that gemcitabine and vinorelbine may be effective agents for patients with lung cancer previously treated with EGFR-TKIs.

Enhanced functional properties of human limbal stem cells by inhibition of the miR-31/FIH-1/P21 axis.

On the basis of the functional roles of the embryonic stem cell niche (ESCN) in the human limbal stem cells (LSCs), we proposed to explore the potential roles of microRNAs in regulating the self-renewal and differentiation of LSCs cultured in the ESCN. The LSCs were cultured in different media, either in CnT-20 media or in CnT-20+20% ES culture supernatant (ESC-CM). The LSCs cultured in ESC-CM were then transfected with microRNA-31 (miR-31) mimic or antago-31. The colony-forming efficiency (CFE) was analysed. Cell cycle, apoptosis, mitochondrial potential and reactive oxygen species were analysed by flow cytometry, and quantitative real-time PCR was used to determine the expression levels of FIH-1, P21, P63, ABCG2, CK3, microRNA-31, microRNA-143, microRNA-145 and microRNA-184. Indirect immunostaining was employed to detect the expression of P63, ABCG2, survivin, connexin-43 and CK3. Western blot was employed to detect the expression of FIH-1, P63, P21, CK3, caspase 3, Tcf4, β-catenin, survivin, GSK3β and pGSK3β. Compared with cells grown in CnT-20, the level of miR-31 in cells grown in ESC-CM was lower. We investigated the roles that miR-31 and FIH-1 play in regulating the functional properties of LSCs. We used antagomirs (antago) to reduce the level of miR-31 in LSCs. Antago-31 increased FIH-1 levels and significantly reduced P21 expressional level in LSCs compared to irrelevant-antago (Ir-antago) treatment. The downregulation of miR-31 in LSCs promotes the maintenance of stemness. ES culture supernatant (ESC-CM) regulates the fate of LSCs in part by inhibiting the miR-31/FIH-1/P21 axis. This study may have a high impact on the expansion of LSCs in regenerative medicine, especially for ocular surface reconstruction.

Microarray-based detection and expression analysis of new genes associated with drug resistance in ovarian cancer cell lines.

PurposeThe present study is to discover a new genes associated with drug resistance development in ovarian cancer.MethodsWe used microarray analysis to determine alterations in the level of expression of genes in cisplatin- (CisPt), doxorubicin- (Dox), topotecan- (Top), and paclitaxel- (Pac) resistant variants of W1 and A2780 ovarian cancer cell lines. Immunohistochemistry assay was used to determine protein expression in ovarian cancer patients.ResultsWe observed alterations in the expression of 22 genes that were common to all three cell lines that were resistant to the same cytostatic drug. The level of expression of 13 genes was upregulated and that of nine genes was downregulated. In the CisPt-resistant cell line, we observed downregulated expression of ABCC6, BST2, ERAP2 and MCTP1; in the Pac-resistant cell line, we observe upregulated expression of ABCB1, EPHA7 and RUNDC3B and downregulated expression of LIPG, MCTP1, NSBP1, PCDH9, PTPRK and SEMA3A. The expression levels of three genes, ABCB1, ABCB4 and IFI16, were upregulated in the Dox-resistant cell lines. In the Top-resistant cell lines, we observed increased expression levels of ABCG2, HERC5, IFIH1, MYOT, S100A3, SAMD4A, SPP1 and TGFBI and decreased expression levels of MCTP1 and PTPRK. The expression of EPHA7, IFI16, SPP1 and TGFBI was confirmed at protein level in analyzed ovarian cancer patients..ConclusionsThe expression profiles of the investigated cell lines indicated that new candidate genes are related to the development of resistance to the cytostatic drugs that are used in first- and second-line chemotherapy of ovarian cancer.

Expression of oxysterol pathway genes in oestrogen-positive breast carcinomas.

This study investigated whether gene expression levels of key modulators of the oxysterol signalling pathway modify the prognosis of patients with oestrogen receptor-positive (ER+) breast carcinomas via interaction with endocrine therapy. The prognosis of patients with ER+ breast carcinoma depends on several factors. Previous studies have suggested that some oxygenated forms of cholesterol (oxysterols) bind to oestrogen receptor and anti-oestrogen binding site which may deregulate cholesterol homoeostasis and influence effect of therapy. The expression levels of 70 oxysterol pathway genes were evaluated in a test set of breast carcinomas differing in ER expression. The genes differentially expressed in ER+ tumours were assessed in a comprehensive set of ER+ tumours to evaluate their clinical significance. A total of 193 primary patients with breast carcinoma were included. The transcript levels were determined by quantitative real-time polymerase chain reaction. The expression levels of 23 genes were found to be specifically dysregulated in ER+ tumours compared to ER- tumours of the test set. The expression levels of ABCG2, CYP7B1, CYP24A1, CYP39A1 and CH25H genes were found to be strongly associated with disease stage; however, none of the gene expression levels were associated with disease-free survival in patients treated with endocrine therapy. The expression of a number of oxysterol pathway genes is significantly modulated by ER expression and associated with the clinical stage of patients. However, the expression of oxysterol pathway genes was not found to modify the prognosis of ER+ patients with breast carcinoma treated with endocrine therapy.

NANOG overexpression and its correlation with stem cell and differentiation markers in meningiomas of different WHO grades.

NANOG, as a key regulator of pluripotency and acting synergistically with other factors, has been described as a crucial transcription factor in various types of cancer. In meningiomas the expression of this marker has not yet been described. With our study, we aimed to identify and localize NANOG and other possible markers of pluripotency, stem cell properties and differentiation in meningioma tissue, to elucidate a possible effect on tumorigenesis. The gene expression levels of NANOG (NANOG1 and NANOGP8), SOX2, OCT4, KLF4, ABCG2, CMYC, MSI1, CD44, NOTCH1, NES, SALL4B, TP53, and EPAS1 were quantitatively examined using RT-qPCR in 33 surgical specimens of low- (WHO grade I) as well as in high-grade (WHO grade II/III) meningiomas with dural tissue as reference. Immunofluorescence co-localization analysis following confocal fluorescence microscopy for NANOG, OCT4, SOX2, Nestin, KI-67, and CD44 was also performed. There was a significant overexpression of NANOG, MSI1, and EPAS1 and a downregulation of NES in all examined tumors. Subgroup analysis (WHO grade I versus grade II/III) revealed differences in the expression of NANOG, CD44, and MSI1. We found 1% NANOG-positive (NANOG+) cells in low-grade and 2% in grade II/III meningiomas co-expressing the other mentioned markers in various compositions. In particular, NANOG+ cells expressing SOX2 and OCT4 were successfully identified (26% low-grade versus 20% high-grade). Our data reveal an overexpression of NANOG and other markers of pluripotency and stemness in meningiomas. Such potentially pluripotent "stem cell-like" cells may have an impact on tumorigenesis and progression in human meningiomas.