Cell Sublines Research Articles

Long Noncoding RNA KIAA0125 Potentiates Cell Migration and Invasion in Gallbladder Cancer.

Gallbladder cancer (GBC) is one of the mostly aggressive diseases with poor prognosis due to the lack of severe symptoms. To date, little is known about the potential roles and underlying mechanisms of long noncoding RNAs (lncRNAs) in GBC initiation and progression. Thus, it provides us with a novel insight into the contribution of lncRNAs to GBC development. Remarkably, we found the differential expression of a lncRNA, namely, KIAA0125, in a pair of GBC cell sublines which possess different metastatic potentials. Then the effects of KIAA0125 on GBC cell migration, invasion, and epithelial-mesenchymal transitions (EMT) were investigated by using a lentivirus-mediated RNA interference (RNAi) system. Notably, cell migration and invasion were strongly inhibited by KIAA0125 suppression. Moreover, the expression of β-Catenin was increased and the expression of Vimentin was decreased in GBC-SD/M cells after KIAA0125 knockdown. Thus, our findings suggested that KIAA0125 promoted the migration and invasion of GBC cells and could serve as a potential therapeutic target in advanced GBC.

MiR-34a inhibits proliferation and invasion of bladder cancer cells by targeting orphan nuclear receptor HNF4G.

miR-34a is a member of the miR-34 family and acts as a tumor suppressor in bladder cancer. This study explored the regulative role of miR-34a on an orphan nuclear receptor HNF4G, which has a well-confirmed role in bladder tumor growth and invasion. qRT-PCR analysis was applied to measure miR-34a expression in two tumorigenic bladder cancer cell lines 5637 and T24 and one normal human urothelial cell line SV-HUC-1. Luciferase assay was performed to verify the putative binding between miR-34a and HNF4G. The influence of miR-34a-HNF4G axis on cell viability, colony formation, and invasion was assessed with loss- and gain-of-function analysis. This study observed that the miR-34a expressions in 5637 and T24 cells were significantly lower than in SV-HUC-1, while the muscle invasive cell sublines 5637-M and T24-M had even lower miR-34a expression than in the nonmuscle invasive sublines. HNF4G has a 3′-UTR binding site with miR-34a and is a direct downstream target of miR-34a. miR-34a can directly downregulate the expression of HNF4G and thus inhibit tumor cell viability, colony formation, and invasion. Therefore, miR-34a-HNF4G axis is an important pathway modulating cell viability, proliferation, and invasion of bladder cancer cells.

Effect of the knockdown of death-associated protein 1 expression on cell adhesion, growth and migration in breast cancer cells.

Death-associated protein 1 (DAP1) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP1 expression and clinicopathological parameters of human breast cancer. In the present study, we aimed to determine the role of DAP1 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sublines of MCF7 and MDA-MB‑231, and performed growth, adhesion and invasion assays and electric cell-substrate impedance sensing (ECIS) studies of the post-wound migration of cells. In addition, we studied the mRNA expression of caspase 8 and 9, DELE, IPS1, cyclin D1 and p21 in the control and knockdown sublines. Knockdown was associated with increased adhesion and migration, significantly so in the MDA-MB-231DAP1kd cell subline (p=0.029 and p=0.001, respectively). Growth in MCF7 cells showed a significant suppression on day 3 (p=0.029), followed by an increase in growth matching the controls on day 5. While no change in the apoptotic response to serum starvation could be attributed to DAP1 knockdown, the expression of known components of the apoptosis pathway (caspase 8) and cell cycle (p21) was significantly reduced in the MCF7DAP1kd cell subline (p≤0.05), while in MDA-MB-231DAP1kd the expression of a pro-apoptotic molecule, IPS1, was suppressed (p≤0.05). DAP1 may have an important role in cell adhesion, migration and growth in the context of breast cancer and has significant associations with the apoptosis pathway. Furthermore, we believe that delayed increase in growth observed in the MCF7DAP1kd cell subline may indicate activation of a strongly pro-oncogenic pathway downstream of DAP1.

Establishment and biological characterization of gallbladder carcinoma cell sublines with higher metastatic potential ability

Objective To establish high transfer potential,high attack ability gallbladder carcinoma cell line (GBC-SD) subline and study its biological characteristics.Methods using Transwell chamber from gallbladder carcinoma cell line (GBC-SD) screening high transfer invasion ability gallbladder carcinoma subline GBC-SD-I-2,then compare cells form; Clone formation test and four methyl azo azole blue (MTT) method to detect the change of cell proliferation ability; Real-time polymerase chain reaction (Real-time PCR) and Western blotting method detection and epithelial mesenchymal transition (EMT) related N-cadherin,β-catenin protein expression change.Results The use of small room Transwell invasion from gallbladder carcinoma cell line were high transfer potential cell subsets and maternal form there are significant differences.Clone formation assay (0.711 ± 0.021 vs.0.445 ± 0.017,P ＜ 0.01),MTT method shows subline proliferation ability noticeably (P ＜ 0.05).Real-time PCR test showed that the group of cells N-cadherin expression level increased significantly (0.881 ± 0.018 vs.0.102 ± 0.020,P ＜ 0.01),β-catenin protein expression decreased obviously (0.625 ± 0.041 vs.0.275 ±0.052,P ＜ 0.05).Western blotting method showed N-cadherin expression level increased significantly (0.758 ± 0.012 vs.0.156 ± 0.011,P ＜ 0.01) ; β-catenin protein expression decreased obviously (0.243 ± 0.014,0.720 ± 0.011,P ＜ 0.01).Conclusion Screening gallbladder carcinoma cell subsets GBC-SD-I-2 attacks,transfer ability enhanced obviously,to explore the gallbladder carcinoma transfer mechanism provides the ideal experimental tools. Key words: Gallbladder neoplasms; Cell line; Neoplasm metastasis

Loss of function of SWI/SNF chromatin remodeling genes leads to genome instability of human lung cancer.

SWI/SNF chromatin remodeling complexes are frequently mutated in a variety of human cancers. We investigated the mutation incidence and the role of mSWI/SNF (BAF) complexes in human lung cancer. In the present study, we analyzed somatic mutations of BAF complexes and other driver mutated genes of lung carcinoma deposited in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. BAF complexes were mutated in 282 of 803 (35.12%) lung carcinoma samples analyzed, ranking second to TP53. Significantly, BAF-mutated samples exhibited more genomic mutations than BAF wild-type ones. Moreover, a significant positive correlation existed between the BAF mutations and overall genomic mutations in these lung carcinoma samples (P<0.001, Pearson's correlation analysis). Specifically, the mutant-typing of 6 BAF genes, SMARCA4, ARID2, ARID1B, BCL11A, BCL11B and BRD9 was associated with more overall mutations in the lung carcinoma samples. A mutation reporter system was developed by means of the establishment of stable cell sublines with slippage-luciferase transcript in a lung adenocarcinoma cell line, Calu-3. SMARCA4, the most frequently mutated BAF gene in lung cancer, was stably knocked down by pSUPER constructs carrying short hairpin RNA (shRNA). Mutation ratios determined from the mutation reporters of Calu-3 cells were significantly increased upon stable SMARCA4 knockdown. We demonstrated that genetic mutations of BAF complexes lead to genome instability of lung carcinoma. Therefore, BAF complexes play an important role in maintaining genome stability in human lung cancer.

Abstract 2110: Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7

Abstract mTOR inhibitor everolimus has been considered as a promising agent for breast cancer treatment especially among estrogen receptor (ER) positive postmenopausal advanced breast cancer. As expected, combination therapy of everolimus and steroidal aromatase inhibitor (AI) showed remarkable clinical efficacy for the first line nonsteroidal AI treatment failure patients in BOLERO-2 trial. However, the subsequent treatment after refractory to that therapy remains a great matter. We already reported several types of long term estrogen-depletion resistant (EDR) cell lines from MCF-7-E10, which has been constitutively ERE-GFP-transfected to monitor ER activity in living cells (Fujiki N et al., J Steroid Biochem Mol Biol. 2013 Oct 13;139C:61-72). They mimic AI resistance and these EDR cells have been characterized as the followings. 1) Type 1 as ER positive (ERE-GFP positive), constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 as ER negative conversion (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated. Then with those two types of sub-cell lines, we generated everolimus resistant cells after long term exposure to everolimus in estrogen depleted medium. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy of AI and everolimus as the second line endocrine treatment after the first line AI treatment failure. Though everolimus extremely inhibited cell proliferation of two EDR cell lines, everolimus resistant cells named as Type 1-EVR, Type 2-EVR no longer responded to everolimus. ER expression and estrogen sensitivity of two types of EVR cells were similar to those of the parental cells, respectively. Pure anti-estrogen fulvestrant inhibited cell proliferation of Type1-EVR cells. On the other hand, Type2-EVR did not respond to fulvestrant but JNK inhibitor inhibited the cell growth. Phosphorylation status of p70S6K and 4E-BP1 were different among individual parental EDR cells and EVR cells. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than the parental cells. In contrast, Type2-EVR expressed phosphorylation of MAP-kinase less than the parental cells. As above, the mechanism of acquired resistance to everolimus was likely to be different between those two sub-cell lines. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance. Citation Format: Mariko Kimura, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Itaru Endo, Shin-ichi Hayashi. Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2014-2110

Abstract 4438: Changes in Nrdp1 regulation of ErbB3 in androgen-dependent vs. independent prostate cancer

Abstract In prostate cancer, the androgen receptor (AR) is a major regulator of gene transcription. We previously demonstrated that the E3 ubiquitin ligase Nrdp1 is transcriptionally regulated by the AR [Chen, L., Cancer Res, 2010]. The effect of the AR on ErbB3 is mediated by Nrdp1, which regulates the degradation of ErbB3. Therefore, we investigated the function of the various domains of Nrdp1 on ErbB3 in prostate cancer. ErbB3 is a major factor in cell growth and proliferation so finding ways to regulate it would be beneficial for patient survival. We found that the Nrdp1 protein had two splice variants, a full length 36kDa protein and a C-terminal 28kDa protein. In addition, the full-length protein remained in the cytoplasm of LNCaP cells, which are androgen dependent, while the 28kDa protein went into the nucleus of C4-2 cells, an androgen independent subline of LNCaP cells. To further investigate the subcellular difference and role of these two splice variants on ErbB3, we used 4 flag tagged vectors that contain either full-length Nrdp1 (Nrdp1), an N-terminal Nrdp1 containing amino acids 1-169 (Zn), a C-terminal Nrdp1 containing amino acids 169-317 (32) , and an Nrdp1 lacking the coiled coil domain (ΔCC). We found that the ΔCC vector caused an increase of cell proliferation in LNCaP cells, an androgen dependent cell line, with no to little change in cell proliferation in its androgen independent sub cell lines, LNCaP R273H (LNCaP stably transfected with a mutated p53), C4-2, and C4-2B, and PC3 and PC3 WT-AR cells. Despite this, we found that the ΔCC protein caused a decrease of ErbB3 protein in all cell lines. We also performed subcellular fractionation on these cell lines transfected with the Nrdp1 flag vectors. All of the Nrdp1 proteins were found in the cytoplasm of the cell lines. In addition, the ΔCC protein was also found in the nuclear fraction of LNCaP R273H, C4-2, and C4-2B, while in LNCaP cells the Zn protein was also found in the nuclear fraction and in PC3 cells the 32 protein was also found in the nuclear fraction. In LNCaP cells, ErbB3 was found in the nucleus when transfected with Nrdp1 protein, Zn protein, and 32 protein, but found in the cytoplasm when transfected with the ΔCC protein. However, in LNCaP R273H, C4-2, and C4-2B cell lines, ErbB3 was found in the nucleus when transfected with all of the Nrdp1 vectors. Based on available data, we concluded that the coiled coil domain is important for ErbB3 protein regulation, but it had opposing effects in castration sensitive and resistant cells. In castration sensitive LNCaP cells, the coiled coil domain of Nrdp1 likely suppresses ErbB3-mediated cell proliferation by preventing ErbB3 translocation from the nucleus to the cytoplasm. However, in castration resistant cells, the coiled coil domain prevents the degradation of ErbB3 by Nrdp1. Further studies are required to determine the mechanism of action of the Nrdp1 coiled coil domain in these two phenotypes. Citation Format: Rosalinda M. Savoy, Salma Siddiqui, William H. Fry, Kermit L. Carraway, Paramita Ghosh. Changes in Nrdp1 regulation of ErbB3 in androgen-dependent vs. independent prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4438. doi:10.1158/1538-7445.AM2014-4438

Long noncoding RNA MALAT1 promotes brain metastasis by inducing epithelial-mesenchymal transition in lung cancer.

Brain metastasis often has a poor prognosis in patients with advanced non-small cell lung cancer (NSCLC). Therefore, it is urgent to identify factors associated with lung cancer brain metastasis. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) also known as noncoding nuclear-enriched abundant transcript 2 is a long noncoding RNA, which is highly conserved amongst mammals. It has been shown to be increased in a variety of tumors including NSCLC and regulate the expression of metastasis-associated genes. However, the role of MALAT1 in lung cancer brain metastasis has not been investigated. In this study, we examined the level of MALAT1 in 78 cases of NSCLC samples with 19 brain metastasis and 59 non-brain metastasis by qRT-PCR. We observed that the level of MALAT1 was significantly higher in brain metastasis than that of non brain metastasis samples (P < 0.001). The level of MALAT1 was associated with patients' survival. To investigate the role of MALAT1 in brain metastasis, we established a highly invasive and metastatic cell subline using the brain metastasis lung cancer cell H1915. We found that MALAT1 is increased in highly invasive subline of brain metastasis lung cancer cells. Further functional studies indicate that silencing MALAT1 inhibits highly invasive subline of brain metastasis lung cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). Therefore, increased level of long noncoding RNA MALAT1 promotes lung cancer brain metastasis by inducing EMT, which may be a promising prognosis factor and therapeutic target to treat lung cancer brain metastasis in future.

Long noncoding RNA MRUL promotes ABCB1 expression in multidrug-resistant gastric cancer cell sublines.

Multidrug resistance (MDR) is the most common cause of chemotherapy failure in gastric cancer (GC) treatment; however, the underlying molecular mechanisms remain elusive. Long noncoding RNAs (lncRNAs) can be involved in carcinogenesis, but the effects of lncRNAs on MDR are poorly understood. We show here that the lncRNA MRUL (MDR-related and upregulated lncRNA), located 400 kb downstream of ABCB1 (ATP-binding cassette, subfamily B, member 1), was significantly upregulated in two multidrug-resistant GC cell sublines, SGC7901/ADR and SGC7901/VCR. Furthermore, the relative expression levels of MRUL in GC tissues were negatively correlated with in vitro growth inhibition rates of GC specimens treated with chemotherapeutic drugs and indicated a poor prognosis for GC patients. MRUL knockdown in SGC7901/ADR and SGC7901/VCR cells led to increased rates of apoptosis, increased accumulation, and reduced doxorubicin (Adriamycin [ADR]) release in the presence of ADR or vincristine. Moreover, MRUL depletion reduced ABCB1 mRNA levels in a dose- and time-dependent manner. Heterologous luciferase reporter assays demonstrated that MRUL might positively affect ABCB1 expression in an orientation- and position-independent manner. Our findings indicate that MRUL promotes ABCB1 expression and is a potential target to reverse the MDR phenotype of GC MDR cell sublines.

BaxΔ2 promotes apoptosis through caspase-8 activation in microsatellite-unstable colon cancer.

Loss of apoptotic Bax due to microsatellite mutation contributes to tumor development and chemoresistance. Recently, a Bax microsatellite mutation was uncovered in combination with a specific alternative splicing event that could generate a unique Bax isoform (BaxΔ2) in otherwise Bax-negative cells. Like the prototype Baxα, BaxΔ2 is a potent proapoptotic molecule. However, the proapoptotic mechanism and therapeutic implication of BaxΔ2 remain elusive. Here, the isolation and analysis of isogenic subcell lines are described that represent different Bax microsatellite statuses from colorectal cancer. Colon cancer cells harboring Bax microsatellite G7/G7 alleles are capable of producing low levels of endogenous BaxΔ2 transcripts and proteins. Interestingly, BaxΔ2-positive cells are selectively sensitive to a subgroup of chemotherapeutics compared with BaxΔ2-negative cells. Unlike other Bax isoforms, BaxΔ2 recruits caspase-8 into the proximity for activation, and the latter, in turn, activates caspase-3 and apoptosis independent of the mitochondrial pathway. These data suggest that the expression of BaxΔ2 may provide alternative apoptotic and chemotherapeutic advantages for Bax-negative tumors. "Bax-negative" colorectal tumors expressing a Bax isoform are sensitive to selective chemotherapeutics.

Establishment and partial characterization of an epirubicin-resistant gastric cancer cell line with upregulated ABCB1.

Multidrug resistance (MDR) is a major impediment to successful chemotherapy of gastric cancer. Our aim was to establish an epirubicin-resistant cell subline (AGS/EPI) and to elucidate the mechanisms involved in acquired EPI resistance. The AGS/EPI cell subline developed by exposing parental AGS cells to stepwise increasing concentrations of EPI demonstrated 2.52-fold resistance relative to the AGS cell line, and mRNA expression of the ATP-dependent drug-efflux pump P-glycoprotein (Pgp), more recently known as ABCB1 protein, was similarly upregulated. An AGS/EPI cell subline could thus be effectively established, and MDR mechanism of these cells was shown to be related to the overexpression of mRNA of the ABCB1 gene.

Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma

Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs.In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP.It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance.

Specific antioxidant compounds differentially modulate cytotoxic activity of doxorubicin and cisplatin: in vitro and in vivo study.

AimTo use the antioxidant compounds (sodium selenite, selenomethionine, D-pantethine) for modulation of cytotoxic effect of doxorubicin and cisplatin toward wild type and drug-resistant mutants of several human tumor cells. Similar treatments were applied in vivo toward adult male Wistar rats.MethodsHuman tumor cells of different lines (HCT-116, Jurkat and HL-60) with various mechanisms of drug-resistance were treated with doxorubicin or cisplatin, alone or in combination with sodium selenite, selenomethionine, or D-pantethine. Cell viability, induction of apoptosis, and production of O2- radicals were measured. Activity of redox potential modulating enzymes was measured in the liver and blood plasma of adult male Wistar rats subjected to similar treatments.ResultsAll antioxidants used in physiologically harmless concentration inhibited cytotoxic action of doxorubicin toward tumor cells sensitive to chemotherapy treatment by 15%-30%, and slightly enhanced cytotoxic effect of this medicine toward drug-resistant malignant cells. At the same time, there was no significant effect of these antioxidants on cisplatin action. Such effects were accompanied by a complete inhibition of production of superoxide radicals induced by doxorubicin. The results of in vivo study in adult male Wistar rats were in agreement with the results of in vitro study of human tumor cells.ConclusionProtective effect of specific antioxidant agents during cytotoxic action of doxorubicin was demonstrated in vitro in drug-sensitive human tumor cells and in adult male Wistar rats, while there was no protective effect in drug-resistant sub-lines of these tumor cells during action of doxorubicin and cisplatin.

Establishment and analysis of osteosarcoma cell sublines with different metastatic characteristics.

The aim of this study is to establish human osteosarcoma MG-63 cell sublines with different metastatic characteristics and provide good experimental models for mechanism study of osteosarcoma metastasis. Six cell sublines were screened and established by using the in vitro cloning technology. In vitro invasion experiments, cellular electrophoretic mobility determinations, cellular proliferation rate determinations, and soft agar clone formation assays were used to compare, analyze, and identify the metastatic characteristics of various cell sublines. The results indicated that the invasion capacity, cellular electrophoretic mobility, cell proliferation, and soft agar clone formation capacity of A2, A3, and A16 sublines were higher than those of A1, A6, and A20 sublines. There was no significant difference in various values determined among A2, A3, and A16, and among A1, A6, and A20 (P > 0.05), but there were significant differences in various values determined between the former three and the latter three (P < 0.05). In conclusion, MG-63 cell sublines with different metastatic characteristics can be established by combining several technologies and can contribute to further research on the mechanism of osteosarcoma metastasis.

Prevention of multidrug resistance (MDR) in osteosarcoma by NSC23925

Background:The major limitation to the success of chemotherapy in osteosarcoma is the development of multidrug resistance (MDR). Preventing the emergence of MDR during chemotherapy treatment has been a high priority of clinical and investigational oncology, but it remains an elusive goal. The NSC23925 has recently been identified as a novel and potent MDR reversal agent. However, whether NSC23925 can prevent the development of MDR in cancer is unknown. Therefore, this study aims to evaluate the effects of NSC23925 on prevention of the development of MDR in osteosarcoma.Methods:Human osteosarcoma cell lines U-2OS and Saos were exposed to increasing concentrations of paclitaxel alone or in combination with NSC23925 for 6 months. Cell sublines selected at different time points were evaluated for their drug sensitivity, drug transporter P-glycoprotein (Pgp) expression and activity.Results:We observed that tumour cells selected with increasing concentrations of paclitaxel alone developed MDR with resistance to paclitaxel and other Pgp substrates, whereas cells cultured with paclitaxel–NSC23925 did not develop MDR and cells remained sensitive to chemotherapeutic agents. Paclitaxel-resistant cells showed high expression and activity of the Pgp, whereas paclitaxel–NSC23925-treated cells did not express Pgp. No changes in IC50 and Pgp expression and activity were observed in cells grown with the NSC23925 alone.Conclusions:Our findings suggest that NSC23925 may prevent the development of MDR by specifically preventing the overexpression of Pgp. Given the significant incidence of MDR in osteosarcoma and the lack of effective agents for prevention of MDR, NSC23925 and derivatives hold the potential to improve the outcome of cancer patients with poor prognosis due to drug resistance.

A new nanostructured carrier design including oil to enhance the pharmaceutical properties of retinoid therapy and its therapeutic effects on chemo-resistant ovarian cancer

All-trans retinoic acid (ATRA) is an appealing alternative drug for the cancers that have failed the conventional chemotherapy and become chemo-resistant and more tumorigenic. In this study, we specifically addressed two issues commonly associated with ATRA nanotherapeutics: (1) insufficient, unstable entrapment and uncontrolled release of the highly lipophilic ATRA and (2) lack of studies in therapeutically relevant chemo-resistant cancer cell models. A polymer-oil nanostructured carrier (PONC) composed of oil and PLGA was designed and studied in an ovarian cancer cell subline SKOV-3PR that could withstand up to 300nM paclitaxel and expressed high levels of multidrug resistance transporter ABCB1 and tumorigenic marker CD133. Differential scanning calorimetry of PONC revealed superior polymer amorphosity and dispersion of the entrapped ATRA in a manner comparable to nanostructured lipid carriers. With this design, the ATRA encapsulation efficiency was increased up to 8.5-fold and a 5-day controlled release profile was obtained. ATRA-PONC was able to induce extensive apoptotic cell death and exert substantially higher long-term anti-tumorigenic effects (IC50 of ATRA-PONC: 2μg/ml versus free ATRA: 17.5μg/ml; p<0.05) in SKOV-3PR cells. Mechanistic studies indicated that these enhanced anticancer effects were likely attributable to higher cell permeation by the well-dispersed drug/oil steadily released from PONC. To conclude, a nanostructured, oil-in-polymer hybrid carrier design has been developed for efficient ATRA delivery and treatment of the chemo-exposed, chemo-resistant sub-population of ovarian cancer, exemplifying a convenient strategy to vastly improve the pharmaceutical and therapeutic properties of tough-to-deliver lipophilic, poorly water-soluble anticancer compounds.

P0222 Stem cell markers predict oral cancer radioresistance

Background Radiotherapy is an important modality in treating head and neck cancers. Clinically, predicting radiation response is essential. Stem-like cancer cells have been reported as having a high degree of radiation resistance. However, whether stem cell markers, such as OCT4 and NANOG, can predict clinical outcomes in patients with resected-then-irradiated oral cancer is still unknown. Methods We established a radioresistant cell subline, OML1_R, from OML-1 oral cancer cells by using sequent 5-Gy irradiations, with a total dose of 50 Gy. Between each irradiation, cancer cells were allowed to regrow for 5–7 days, then PCR was used to detect mRNA expression of stem cell markers. Paraffin-embedded human tissue sections were used for validation of the role of stem cell markers in predicting post-resection, post-irradiation clinical outcomes. Findings A 10-Gy radiation stress test confirmed the radioresistance of OML1_R. PCR showed upregulation of OCT4 (4-fold) and NANOG (6-fold) in OML1_R cells compared with their parent OML-1 cells. In 44 patients with resected-but-close-margined oral cancer, higher locoregional control was observed in the low OCT4/NANOG expression group compared with the high OCT4/NANOG expression group (92% versus 62%, p = 0.03). Multivariate analysis confirmed that this stem cell marker is an independent factor in predicting locoregional control (adjusted hazard ratio 6.76; 95% confidence interval 1.87–24.43; p = 0.04). Interpretation Our data showed that stem cell markers OCT4 and NANOG are potential bio-predictors in patients with resected-then-irradiated oral cancer.

FTSJ2, a Heat Shock-Inducible Mitochondrial Protein, Suppresses Cell Invasion and Migration

Ribosomal RNA large subunit methyltransferase J (RrmJ), an Escherichia coli heat shock protein, is responsible for 2′-O-ribose methylation in 23S rRNA. In mammals, three close homologs of RrmJ have been identified and have been designated as FTSJ1, FTSJ2 and FTSJ3; however, little is known about these genes. In this study, we characterized the mammalian FTSJ2, which was the most related protein to RrmJ in a phylogenetic analysis that had similar amino acid sequence features and tertiary protein structures of RrmJ. FTSJ2 was first identified in this study as a nucleus encoded mitochondrial protein that preserves the heat shock protein character in mammals in which the mRNA expressions was increased in porcine lung tissues and A549 cells after heat shock treatment. In addition, a recent study in non-small cell lung cancer (NSCLC) suggested that the FTSJ2 gene is located in a novel oncogenic locus. However, our results demonstrate that the expression of FTSJ2 mRNA was decreased in the more invasive subline (CL1-5) of the lung adenocarcinoma cells (CL1) compared with the less invasive subline (CL1-0), and overexpression of FTSJ2 resulted in the inhibition of cell invasion and migration in the rhabdomyosarcoma cell (TE671). In conclusion, our findings indicate that mammalian FTSJ2 is a mitochondrial ortholog of E. coli RrmJ and conserves the heat shock protein properties. Moreover, FTSJ2 possesses suppressive effects on the invasion and migration of cancer cells.

Nuclear factor-ĸB plays a critical role in both intrinsic and acquired resistance against endocrine therapy in human breast cancer cells

Since more than 75% of breast cancers overexpress estrogen receptors (ER), endocrine therapy targeting ER has significantly improved the survival rate. Nonetheless, breast cancer still afflicts women worldwide and the major problem behind it is resistance to endocrine therapy. We have previously shown the involvement of nuclear factor-κB (NF-κB) in neoplastic proliferation of human breast cancer cells; however, the association with the transformation of ER-positive cells remains unclear. In the current study, we focused on roles of NF-κB in the hormone dependency of breast cancers by means of ER-positive MCF-7 cells. Blocking of NF-κB signals in ER-negative cells stopped proliferation by downregulation of D-type cyclins. In contrast, the MCF-7 cells were resistant to NF-κB inhibition. Under estrogen-free conditions, the ER levels were reduced when compared with the original MCF-7 cells and the established cell subline exhibited tamoxifen resistance. Additionally, NF-κB participated in cell growth instead of the estrogen-ER axis in the subline and consequently, interfering with the NF-κB signals induced additive anticancer effects with tamoxifen. MMP-9 production responsible for cell migration, as well as the cell expansion in vivo, were suppressed by NF-κB inhibition. Therefore, we suggest that NF-κB is a master switch in both ER-positive and ER-negative breast cancers.

Synergistic effect of ginsenoside Rg3 with verapamil on the modulation of multidrug resistance in human acute myeloid leukemia cells.

The pharmacological modulatory effects of 20(S)-ginsenoside Rg3 (20S-Rg3) on multidrug resistant cancer cells are reported in the present study. The effects of 20(S)-Rg3 on the modulation of doxorubicin (DOX) and vincristine (VCR) resistance were examined in the HL60 multidrug resistant subline of human acute myeloid leukemia cells. Results demonstrated that 20S-Rg3 is as effective as verapamil (Vp) for modulating the high degree primary DOX resistance and low degree VCR cross-resistance expressed by the H160 cell line. Furthermore, the present study demonstrates for the first time, using isobologram analysis, that the combination of 20S-Rg3 and Vp enhances the reversal of DOX and VCR resistance in a supra-additive or at least an additive manner. These results indicate that 20S-Rg3 may be used as a Vp synergizer or as a promising alternative to Vp in the chemosensitization of multidrug resistant acute myeloid leukemia, with far fewer side effects.