Dox In Cells Research Articles

Abstract 3441: Silencing glucosylceramide synthase using MBO-asGCS eliminates tumor metastasis

Abstract Disseminated metastasis is often observed in cancer patients fail in chemotherapy. Tumor metastasis is known to rely on the interaction of cancer cells and the extracellular matrix components but the molecular mechanism by which drug resistance promotes tumor metastasis is still not clear. Glucosylceramide synthase (GCS) that is a limiting enzyme for ceramide glycosylation and regulates glycosphingolipid (GSL) synthesis can cause drug resistance. Hence we report that GCS is a determinant of metastatic properties of drug-resistant cancer cells, and silencing GCS eliminates tumor metastasis. Our recent study found that the metastatic potency of drug-resistant cancer cells depended on the overexpression of GCS. Compared with drug-sensitive MCF-7 cells, the wound healing, matrigel attachment and MMP-9 activity significantly was 1.5 fold faster, 3 fold more and 2 folds higher respectively in the drug resistant MCF-7/Dox that also had 39-fold higher GCS protein level and 2-fold higher GCS enzyme activity. Introduction of GCS gene enhanced the metastatic potency of MCF-7 cells, because its matrigel attachment and invasion potential significantly increased by 2-fold and 1.5-fold after transfection. In contrast, silencing GCS by using mixed-backbone oligonucleotide against GCS (MBO-asGCS, 100 nM) substantially diminished the metastatic potency of MCF-7/Dox cells, as the wound healing, matrigel attachment and invasion reduced by 2-fold, 1.5-fold and 3-fold respectively. MBO-asGCS treatments eliminated lung metastasis of MCF-7/Dox tumors. Further mechanistic studies disclosed that silencing GCS by MBO-asGCS decreased its expression and enzyme activity and also decreased the levels of globo-series glycosphingolipids (GSLs) in GSL-enriched microdomain located on the plasma membrane. Sequentially, silencing GCS decreased the expression levels of uPA, MMP-9, VEGF and FGF-2 that actively mediated the migration and invasion of cancer cells. This study thus indicates that GCS is determinant for the metastatic potency of drug-resistant cancer cells and silencing GCS by using MBO-asGCS is an effective approach to target the metastasis of tumor with drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3441. doi:1538-7445.AM2012-3441

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.

ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Abstract 2745Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210).While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NATo further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM’s (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1.These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures:White:BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Runx2 promotes both osteoblastogenesis and novel osteoclastogenic signals in ST2 mesenchymal progenitor cells

We profiled the global gene expression of a bone marrow-derived mesenchymal pluripotent cell line in response to Runx2 expression. Besides osteoblast differentiation, Runx2 promoted the osteoclastogenesis of co-cultured splenocytes. This was attributable to the upregulation of many novel osteoclastogenic genes and the downregulation of anti-osteoclastogenic genes. In addition to being a master regulator for osteoblast differentiation, Runx2 controls osteoblast-driven osteoclastogenesis. Previous studies profiling gene expression during osteoblast differentiation had limited focus on Runx2 or paid little attention to its role in mediating osteoblast-driven osteoclastogenesis. ST2/Rx2(dox), a bone marrow-derived mesenchymal pluripotent cell line that expresses Runx2 in response to Doxycycline (Dox), was used to profile Runx2-induced gene expression changes. Runx2-induced osteoblast differentiation was assessed based on alkaline phosphatase staining and expression of classical marker genes. Osteoclastogenic potential was evaluated by TRAP staining of osteoclasts that differentiated from primary murine splenocytes co-cultured with the ST2/Rx2(dox) cells. The BeadChip™ platform (Illumina) was used to interrogate genome-wide expression changes in ST2/Rx2(dox) cultures after treatment with Dox or vehicle for 24 or 48h. Expression of selected genes was also measured by RT-qPCR. Dox-mediated Runx2 induction in ST2 cells stimulated their own differentiation along the osteoblast lineage and the differentiation of co-cultured splenocytes into osteoclasts. The latter was attributable to the stimulation of osteoclastogenic genes such as Sema7a, Ltc4s, Efnb1, Apcdd1, and Tnc as well as the inhibition of anti-osteoclastogenic genes such as Tnfrsf11b (OPG), Sema3a, Slco2b1, Ogn, Clec2d (Ocil), Il1rn, and Rspo2. Direct control of osteoblast differentiation and concomitant indirect control of osteoclast differentiation, both through the activity of Runx2 in pre-osteoblasts, constitute a novel mechanism of coordination with a potential crucial role in coupling bone formation and resorption.

Fas/FasL-dependent and -independent activation of caspase-8 in doxorubicin-treated human breast cancer MCF-7 cells: ADAM10 down-regulation activates Fas/FasL signaling pathway

The contribution of Fas-mediated death pathway to doxorubicin-induced death of MCF-7 cells is not unambiguously elucidated. Thus, this study was conducted to explore doxorubicin-induced Fas/FasL signaling pathway activation in MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Dox) cells. Doxorubicin-induced caspase-8 activation was found to be mediated through Akt/ERK inactivation and FasL-independent Fas pathway in MCF-7 cells, while caspase-8 activation in MCF-7/Dox cells depended exclusively on FasL-stimulated Fas pathway. Suppression of caspase-8 activation restored the viability of doxorubicin-treated MCF-7 cells and MCF-7/Dox cells. Contrary to FasL surface expression exclusively detected in MCF-7/Dox cells, intracellular FasL expression was noted with MCF-7 cells. Promotion of FasL translocation to the cell surface by lysophosphatidic acid evoked a FasL-activated Fas death pathway in MCF-7 cells. Doxorubixin-evoked β-TrCP up-regulation promoted Sp1 degradation, which subsequently suppressed ADAM10 expression in MCF-7 and MCF-7/Dox cells. Doxorubicin-induced down-regulation of ADAM10 reduced FasL shedding, leading to Fas pathway activation in MCF-7/Dox cells. Knock-down of ADAM10 induced death in MCF-7/Dox cells, but marginally reduced the viability of MCF-7 cells. Taken together, our data indicate that Akt/ERK-mediated caspase-8 activation and Fas/FasL-mediated caspase-8 activation mostly elucidate doxorubicin-induced death in MCF-7 cells and MCF-7/Dox cells, respectively. These observations suggest a promising therapeutic modality for overcoming doxorubicin-resistant breast cancer by targeting ADAM10 sheddase activity.

Involvement of Cox-2 in the metastatic potential of chemotherapy-resistant breast cancer cells

BackgroundA major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. However, the mechanisms underlying this drug resistance are unknown. To study the molecular mechanisms underlying the invasive and metastatic activities of drug-resistant cancer cells, we generated a doxorubicin-resistant MCF-7 breast cancer cell line (MCF-7/DOX).MethodsWe used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, DNA fragmentation assays, Western blot analysis, cell invasion assays, small interfering RNA (siRNA) transfection, reverse transcription-polymerase chain reaction, experimental lung metastasis models, and gelatin and fibrinogen/plasminogen zymography to study the molecular mechanism of metastatic activities in MCF-7/DOX cells.ResultsWe found that MCF-7/DOX acquired invasive activities. In addition, Western blot analysis showed increased expression of epidermal growth factor receptor (EGFR) and Cox-2 in MCF-7/DOX cells. Inhibition of Cox-2, phosphoinositide 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase (MAPK) pathways effectively inhibited the invasive activities of MCF-7/DOX cells. Gelatin and fibrinogen/plasminogen zymography analysis showed that the enzymatic activities of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator were markedly higher in MCF-7/DOX cells than in the MCF-7 cells. In vitro invasion assays and mouse models of lung metastasis demonstrated that MCF-7/DOX cells acquired invasive abilities. Using siRNAs and agonists specific for prostaglandin E (EP) receptors, we found that EP1 and EP3 played important roles in the invasiveness of MCF-7/DOX cells.ConclusionsWe found that the invasive activity of MCF-7/DOX cells is mediated by Cox-2, which is induced by the EGFR-activated PI3K/Akt and MAPK pathways. In addition, EP1 and EP3 are important in the Cox-2-induced invasion of MCF-7/DOX cells. Therefore, not only Cox-2 but also EP1 and EP3 could be important targets for chemosensitization and inhibition of metastasis in breast cancers that are resistant to chemotherapy.

Tetramethylpyrazine reverses multidrug resistance in breast cancer cells through regulating the expression and function of P-glycoprotein

Tumor multidrug resistance (MDR) has become the major obstacle to cancer chemotherapy. Recent studies suggest that tetramethylpyrazine (TMP) could reverse tumor MDR although the mechanism by which TMP overcomes tumor MDR remains elusive. Therefore, in this study, we examined the effects of TMP on MDR in drug-resistant breast cancer cells and investigated the underlying mechanisms. MCF-7 cells and the derived P-glycoprotein (Pgp) overexpressing MCF-7/dox cells were treated with TMP, and their growth was examined by MTT assay. Doxorubicin accumulation in the cells was evaluated by flow cytometry, and the expression of Pgp was detected by Western blot and RT-PCR analysis. The results showed that TMP increased the intracellular concentration of doxorubicin and inhibited Pgp-mediated efflux of doxorubicin in a dose-dependent manner. Moreover, TMP inhibited the ATPase activity of P-gp and suppressed the expression of Pgp in MCF-7/dox cells. Taken together, these data suggest that TMP has potential application in the treatment of chemotherapy-resistant breast cancer.

Abstract 4365: Glucosylceramide synthase promotes cancer stem cells accumulating in chemotherapy

Abstract Cancer stem cells initiate tumorigenesis and are the causes of metastasis and relapse. Cancer stem cells, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but we do not know whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). We have found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD24-/CD44+/ESA+ phenotype was increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. BCSCs sorted from MCF-7/Dox cells displayed significantly higher GCS enzyme activity more than 3-fold and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells showed significantly higher tumorigenicity and metastasibility as compared to non-stem cells (CD24-/CD44-) in athymic nude mice. More interestingly, doxorubicin treatment (1 mg/kg, once a week, and 40 days) considerably increased BCSCs 2-fold in tumors, and MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs more than 2-fold in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulates FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMAR1, PPARD and downregulates CXCL12, FRAT1, CCND1 to accumulate BCSCs during the course of chemotherapy. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes aggressive tumor progression. Silencing of GCS that eliminates BCSCs can prevent and treat drug-resistant tumors and relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2011-4365

Abstract 1470: Depletion of glycosphingolipids with MBO-asGCS eliminates cancer cell metastasis

Abstract Tumor metastasis, particularly multi-organ metastasis often causes failure of cancer treatment. It is well-known that the interaction of tumor cells with extracellular matrix (ECM) determines the metastatic ability of cancer cells; however, it is not clear whether depletion of glycosphingolipids (GSLs), a major component of GSL-enriched microdomain on plasma membrane is an approach to eliminate tumor metastasis. Glucosylceramide synthase (GCS) that converts ceramide to glucosylceramide is a limiting enzyme controlling GSL synthesis. We found that overexpression of GCS enhanced metastatic ability of human breast cancer cells. In MCF-7/Dox cells, GCS protein levels were increased 39 folds and GCS activity were increased 2 folds, as compared to MCF-7 cells. The wound healing, Matrigel attachment and MMP-9 activity in MCF-7/Dox cells increased 1.5 folds, 3 folds, and 2 folds respectively compared to MCF-7 cells. Introduction of GCS gene overexpressed GCS protein and significantly increased wound healing and Matrigel attachment by 2 folds and cell invasion 1.5 folds in MCF-7 cells. MBO-asGCS is a mixed backbone oligonucleotide that substantially suppresses GCS expression. Silencing of GCS with MBO-asGCS (100 nM) decreased the wound healing by 2 folds, Matrigel attachment by 1.5 folds and cell invasion by 3 folds in MCF/Dox cells. These results were also observed in MCF-7/DOX cells treated with d-PDMP, a GCS inhibitor. Mechanistic study indicated that overexpression of GCS increased GSLs, particular globo-series GSLs and enhanced VEGF and uPA to promote the mobility, cellular attachment and invasion of cancer cells. As GCS overexpression is tightly associated with drug-resistant cancer cells, this study may suggest that depletion of GSLs with MBO-asGCS is an effective approach to eliminate the metastasis of cancers that even fail in conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2011-1470

First characterization of fish P-glycoprotein (abcb1) substrate specificity using determinations of its ATPase activity and calcein-AM assay with PLHC-1/dox cell line

P-glycoprotein (P-gp; abcb1) is one of the major ABC transport proteins that mediates multixenobiotic resistance (MXR) defense in fish. In order to offer a sound evaluation of its ecotoxicological relevance it is critical to characterize substrate specificity of fish P-gp. Measurement of the ATPase activity is a reliable approach often used to discern type of interaction of various drugs with mammalian P-gp. A similar assay has never been used for characterization of P-gp in aquatic organisms and the main goal of this study was to develop a specific ATPase assay for characterization of fish P-gp. For this purpose we have used P-gp enriched membrane vesicles isolated from fish hepatoma PLHC-1/dox cells characterized by high overexpression of P-gp. As additional demonstration of a P-gp specific phenotype, we have quantified transcript expression of a series of eight ABC efflux transporter genes constitutively expressed in PLHC-1 wild type and PLHC-1/dox cells. Transcript expression analysis confirmed high and specific P-gp transcript overexpression in PLHC-1/dox cells. Provided that the transcript abundance is translated to protein, the development of ATPase assay is enabled. Using this model we determined Km ATP of 0.4 mM, baseline ATPase activity from 35–50 nmol/mg PROT/min, and maximal activation of ATPase activity obtained for fish P-gp in our system was 1.8–2.5-fold over baseline. All these values were in good agreement with data previously reported for mammalian P-gp. In order to perform a more detailed characterization of fish P-gp substrate specificity, in the next step of our study we used the developed ATPase assay to test 50 different compounds for their interaction with fish P-gp. The same set of compounds was also tested with calcein-AM (Ca-AM) transport activity assay both using PLHC-1/dox cells and NIH 3T3/MDR1 fibroblast cells overexpressing human P-gp. Our results showed that there is a clear difference for some substances—five compounds specifically interacted only with fish P-gp, while seven compounds exhibited interaction with human P-gp only. Most of the compounds tested in this study showed similar behavior in respect to fish or human P-gp and relatively high correlation in the interaction potency was found between fish and human P-gp. In summary, the described results represent the first in depth insight into substrate specificity of an important xenobiotic efflux transporter in fish. In addition, our study showed that combination of Ca-AM assay and the developed ATPase assay using inside/out vesicles isolated from PLHC-1/dox cells, offers a high-throughput and reliable approach for identification of environmentally relevant pollutants that interact with fish P-gp.

Down-regulation of ICBP90 contributes to doxorubicin resistance

Acquired resistance to doxorubicin has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this has not been completely elucidated. In this study, a doxorubicin-resistant MCF-7/Dox cell was developed to mimic the occurrence of acquired doxorubicin resistance. We next contrasted the expression profiles of ICBP90 and Topo IIα and tumor cell growth of different breast cancer cell lines to doxorubicin. Decreased expression levels of ICBP90 and Topo IIα were found in doxorubicin-resistant cells. To examine its function in chemoresistance, RNA interference (RNAi) and forskolin stimulation experiments further demonstrated that ICBP90 and Topo IIα were involved in the proliferation of cells that had acquired doxorubicin resistance. In MCF-7/Dox and ICBP90-siRNA cells, the cell growth wasn't inhibited by doxorubicin and preferentially arrested in G1 phase. However, after forskolin increased the Topo IIα expression, these breast cancer cells were again found to be inhibited by doxorubicin. Further, immunohistochemical assay breast cancer patients accepted EFC regimen showed ICBP90 was significantly associated with tumor cell proliferation, locally advanced disease and Topo IIα expression. In conclusion, down-regulation of ICBP90 induced the descended expression of Topo IIα protein which is the target enzyme of doxorubicin.

Reversal of Multidrug Resistance by Guggulsterone in Drug-Resistant MCF-7 Cell Lines

Background: Multidrug resistance (MDR) presents a serious problem in cancer chemotherapy. Our previous studies have shown that the MDR of K562/DOX cells could be reversed by guggulsterone through inhibiting the function and expression of P-glycoprotein. The purpose of this study was to investigate the reversal effect of guggulsterone on MDR in drug-resistant MCF-7 cells and the parental MCF-7 cells. Methods: MTT cytotoxicity assays, flow cytometry, and Western blot analysis were performed to investigate the antiproliferative effects of the combination of anticancer drugs with guggulsterone, to study the reversal of drug resistance and to examine the inhibitory effects on MRP1 expression. Results: The results showed that co-administration of guggulsterone (10 µM) resulted in a significant increase in chemosensitivity of MCF-7/DOX cells to doxorubicin, compared with doxorubicin treatment alone (p < 0.01). The fold reversal of 10 µM guggulsterone (11.48) was comparable to that of 10 µM verapamil (13.23). Rhodamine123 and doxorubicin accumulation in MCF-7/DOX cells was significantly enhanced after the incubation with guggulsterone (10 µM), compared with untreated MCF-7/DOX cells (p < 0.01). When doxorubicin (10 µM) was combined with guggulsterone (10 µM), the mean apoptotic population of MCF-7/DOX cells was 24.91%. It was increased by 6.15 times, compared with doxorubicin (10 µM) treatment alone. However, guggulsterone had little inhibitory effect on the expression of MRP1 proteins. Conclusion: Guggulsterone is a novel and potent MDR reversal agent with the potential to be an adjunctive agent for tumor chemotherapy.

Modelling of TKI Resistance In CML Cell Lines: Kinase Domain Mutations Usually Arise In the Setting of BCR-ABL Overexpression.

AbstractAbstract 3383There are three currently identified secondary resistance mechanisms observed in chronic myeloid leukemia (CML) patients receiving tyrosine kinase inhibitors (TKIs). These include overexpression of drug-efflux proteins (ABCB1 and ABCG2), increased BCR-ABL expression, and mutations in the kinase domain (KD) of BCR-ABL. We investigated the interplay between these three modes of resistance in vitro, as well as looking for other mechanisms. Three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant, K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib (IM) to 2μ M, or dasatinib (DAS) to 200nM. Two IM-resistant K562 lines were established, both with increased IC50s for IM (from 13.7μ M in naïve cells, to ~50μ M), as well as increased IC50s for DAS and nilotinib (NIL). No cell-surface expression of ABCB1 or ABCG2 was observed, nor were KD mutations present. However, BCR-ABL expression was seen to steadily increase in both lines from 178% in naïve cells, to ~380% and 1200% in the resistant lines, suggesting this was the major mode of resistance. However, when a DAS-resistant K562 culture was generated the T315I mutation emerged. Studies of the intermediate stages of resistance revealed that BCR-ABL overexpression occurred in a step-wise fashion, peaking at 1915% in the 3.5nM intermediate, but then dropping significantly to ~1000% in the 5nM intermediate (P=0.0003). BCR-ABL expression then stabilised at this level, and the T315I mutation was subsequently detected. Thus, it appears that BCR-ABL overexpression was the first mechanism of resistance detectable, but was followed by the emergence of a KD mutation which had a clear selective advantage. This sequential selection was observed a further four times: in a DAS-resistant K562 Dox culture, and in three IM-resistant KU812 cultures. BCR-ABL expression in the DAS-resistant K562 Dox culture increased from 186% in naïve cells to 540% in the final culture. Studies of intermediate cultures revealed that BCR-ABL expression peaked at 850% in the 55nM intermediate, but then dropped significantly to ~500% in the 75nM intermediate (P=0.004). This drop in BCR-ABL expression coincided with the appearance of the V299L mutation. Interestingly, the K562 Dox DAS-resistant line also displayed resistance to NIL and IM, likely conferred by BCR-ABL overexpression as the 55nM intermediate (with the highest BCR-ABL expression levels) had the highest IC50s for NIL and IM, while the 75nM intermediate (with the V299L mutation) had increased IC50DAS but lower NIL and IM IC50s. Thus, BCR-ABL overexpression was the primary event, followed by the KD mutation. Likewise in three IM-resistant KU812 cultures, BCR-ABL expression levels rose from 443% in naïve cells, to peak levels of 6210%, 10,448% and 990% respectively, followed by drops in expression which coincided with the appearance of compound KD mutations, and the F359C mutation respectively (Table). In contrast, three IM-resistant K562 Dox cells were not found to have any KD mutations, nor BCR-ABL overexpression. Instead, the primary cause of resistance in these lines appears to be a further increase in ABCB1 expression. All three lines had increased IC50s for IM (from 12μ M in naïve cells, to ~27μ M), NIL (from 400nM to ~1000nM) and DAS (from 100nM to >625nM). The addition of PSC833 (an ABCB1 inhibitor) decreased the IM, NIL, and DAS IC50s for all three resistant lines to the level of the naïve control (~3μ M, ~250nM and ~10nM respectively), indicating that ABCB1 expression, facilitating active efflux of the drugs, is the primary mechanism of resistance in these lines. We have demonstrated that KD emergence is a stochastic event, as the same mutation did not always occur twice, however BCR-ABL and ABCB1 overexpression were more likely to arise recurrently in predisposed lines. Notably, different TKIs elicited different resistant mechanisms, but all were BCR-ABL dependent. Furthermore, all resistant lines showed cross-resistance to the three TKIs tested (IM, DAS and NIL), suggesting that currently available TKIs share the same susceptibilities to drug resistance.Table 1Summary of resistance mechanisms detected in three cell lines exposed to IM or DAS. ✓ = yes; × = no.Culture conditionK562K562 DoxKU812IMIMDASIMIMIMDASIMIMIMResistance to 3 TKIs✓✓✓✓✓✓✓✓✓✓KD MutationxxT315IxxxV299LE450QE459KE470KE459KE462KE466EF359CIncreased BCR-ABL✓✓✓xxx✓✓✓✓Increased ABCB1xxx✓✓✓xxxx Disclosures:White:Novartis Pharmaceuticals: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Hughes:Novartis Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

Linoleic acid-grafted chitosan oligosaccharide micelles for intracellular drug delivery and reverse drug resistance of tumor cells

Intracellular drug delivery is an important rout to reverse drug resistance of tumor cells. In this study, the linoleic acid (LA)-grafted chitosan oligosaccharide (CSO) was synthesized to construct a micellar delivery system for intracellular delivery. The synthesized linoleic acid-grafted chitosan oligosaccharide (CSO-LA) with 10.3% graft ratio of LA could form micelles in aqueous with 86.69 μg/ml critical micellar concentration (CMC). The CSO-LA micelle had 46.2 ± 3.6 nm number average diameter and 36.0 ± 3.3 mV zeta potential. Taking doxorubicin base (DOX) as a model drug, the drug-loaded CSO-LA micelles (CSO-LA/DOX) was then prepared. The drug encapsulation efficiencies of CSO-LA/DOX were as high as 80%, and the drug loading capacity could be improved by increasing the charged DOX. Using MCF-7, Doxorubicin·HCl resistant MCF-7 (MCF-7/ADR), K562 and Doxorubicin·HCl resistant K562 (K562/ADR) cells as model drug sensitive and drug resistant tumor cells, the experiments demonstrated the CSO-LA had excellent cellular uptake ability by either drug sensitive tumor cells or drug resistance tumor cells. The CSO-LA micelles could deliver DOX into tumor cells, and the DOX in cells was increased with incubation time. As a result, the cytotoxicities of DOX encapsulated in CSO-LA micelles against drug resistance tumor cells were improved significantly, comparing to that of Doxorubicin·HCl solution.

Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells

The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.

Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF-7 cells

One of the major problems of cancer chemotherapy is the development of multidrug resistance (MDR) phenotype. Among the numerous mechanisms of MDR, a prominent one is the increased expression of membrane transporter proteins, the action of which leads to decreased intracellular drug concentration and cytotoxicity of drugs. Among them, P-gp and MRP1, encoded by MDR1 and MRP1 genes, respectively, have been associated with MDR phenotype. Chemical modulators can be used to reverse MDR. These chemicals can either modulate MDR due to their substrate analogy (such as calcium channel blocker verapamil) or interact with phospholipid membranes (such as antihistaminic drug promethazine). This study focuses on the effect of verapamil and promethazine on the expression levels of MDR1 and MRP1 genes and the drug transport activity in doxorubicin-resistant MCF-7 breast carcinoma cell line. Doxorubicin-resistant MCF-7 (MCF-7/Dox) cells were incubated with either verapamil or promethazine, and total RNA was isolated. Real-time PCR (qPCR) was carried out by using specific primers for MDR1, MRP1, and β-actin genes. Intracellular doxorubicin accumulation was also examined by confocal laser scanning microscopy in treated cells. Results demonstrated a significant decrease in both MDR1 and MRP1 expression levels after promethazine applications. It has also been shown that treatment of the cells with verapamil results in significant decrease in MDR1 mRNA levels. Confocal laser scanning microscopy images demonstrated that the intracellular accumulation of doxorubicin was increased after verapamil treatment in MCF-7/Dox cells. The present study gives an idea about the efficiency of verapamil and promethazine on MDR reversal both in gene expression and in transport activity levels.

TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters

BackgroundExposure of cancer cells to chemotherapeutic agents may result in reduced sensitivity to structurally unrelated agents, a phenomenon known as multidrug resistance, MDR. The purpose of this study is to investigate cell growth inhibition of wild type and the corresponding MDR cells by Tumor Treating Fields - TTFields, a new cancer treatment modality that is free of systemic toxicity. The TTFields were applied alone and in combination with paclitaxel and doxorubicin.MethodsThree pairs of wild type/MDR cell lines, having resistivity resulting from over-expression of ABC transporters, were studied: a clonal derivative (C11) of parental Chinese hamster ovary AA8 cells and their emetine-resistant sub-line EmtR1; human breast cancer cells MCF-7 and their mitoxantrone-resistant sub lines MCF-7/Mx and human breast cancer cells MDA-MB-231 and their doxorubicin resistant MDA-MB-231/Dox cells. TTFields were applied for 72 hours with and without the chemotherapeutic agents. The numbers of viable cells in the treated cultures and the untreated control groups were determined using the XTT assay. Student t-test was applied to asses the significance of the differences between results obtained for each of the three cell pairs.ResultsTTFields caused a similar reduction in the number of viable cells of wild type and MDR cells. Treatments by TTFields/drug combinations resulted in a similar increased reduction in cell survival of wild type and MDR cells. TTFields had no effect on intracellular doxorubicin accumulation in both wild type and MDR cells.ConclusionsThe results indicate that TTFields alone and in combination with paclitaxel and doxorubicin effectively reduce the viability of both wild type and MDR cell sub-lines and thus can potentially be used as an effective treatment of drug resistant tumors.

Abstract 4282: The role of glycosphingolipids in the formation of cancer stem cells and drug resistance

Abstract Tumorigenesis and malignant progression of cancers rely on a small subset of cells known as cancer stem cells (CSCs). In addition to forming primary tumors, metastasis and relapse, CSCs always display resistance to cytotoxins and this resistant feature protects CSCs during the course of chemotherapy. Glucosylceramide synthase (GCS) is a limited enzyme catalyzing the first glycosylation in glycosphingolipids synthesis. Glycosphingolipids are likely to play an essential role in maintaining the stemness of stem cells, since that deletion of GCS induces apoptosis of embryonic stem cell and stops embryo development in GCS knockout mice. The stage-specific embryonic antigens (SSEA-3, SSEA-4) are glycosphingolipids and serve as common pluripotent markers for human embryonic stem cells. In the present study, we have found that GCS modulates the formation and maintenance of breast cancer stem cells. GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs) and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cell. Analyzed by using flow cytometry and immunostaining, the BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other populations sorted from MCF-7/Dox. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs in MCF-7/Dox cells. We observed tumorigenesis of NCI/ADR-RE/GCS (GCS overexpressed) and NCI/ADR-RE/asGCS (GCS silenced) in athymic nude mice. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSC-enriched; however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. Furthermore, MBO-asGCS treatment (1 mg/kg) significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES, as compared with control groups. Furthermore, BCSCs after MBO-asGCS treatment were more sensitive to doxorubicin. These results, for the first time, demonstrate that glycosphingolipid is involved in the regulation of cancer stem cell formation. Silencing GCS eliminates BCSCs, that can reverses drug resistance as well as prevent tumor relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4282.

Abstract 5419: Effects of selective aryl hydrocarbon receptor modulators (SAhRMs) in enhancing activity of tamoxifen against doxorubicin resistant MCF-7 Cells: Role of BRCA1

Abstract Recent reports indicate that SAhRMs such as diindolylmethane (DIM) inhibit α-estrogen receptor (ER) expression and estrogen signaling in human breast cancer cells. There is also increasing evidence that SAhRMs demonstrate anti estrogenic and antitumorigenic activity in ER positive human breast adenocarcinoma (MCF-7) cells via cross talk between aromatic hydrocarbon receptor (AhR) and ER pathways. Additional reports suggest that BRCA1 transcriptional activity is enhanced by interactions with AhR and the levels of BRCA1 expression play a role in tamoxifen sensitivity/resistance in breast cancer cells by altering estrogen receptor-coregulator interactions. However, the effects of AhR and its agonists and antagonists, and their modulatory effect on BRCA1 in resistant human breast cancer cells have not been investigated. We have tested the hypothesis that DIM, an AhR agonist may inhibit cell growth in a concentration and time dependant manner against doxorubicin resistant MCF-7 (MCF-7/dox) cells and may enhance the activity of 4-OH tamoxifen (TAM) against both MCF-7 and MCF-7/dox cells by enhancing the expression of BRCA1. Initially we determined the cytotoxic effect of DIM against the two cell lines by utilizing the MTT assay. Both DIM and TAM were equally effective against MCF-7 and MCF-7/dox cells with the IC50 values of approximately 90 μM and 20 μM, respectively at 72 hr. However, a combination of DIM and TAM resulted in an increase in cytotoxcity and statistically significant lowering of the IC50 concentration to 50 μM and 10 μM for each drug, respectively. We also investigated the effects of AhR antagonist, α-naphthoflavone (NF) which had a similar IC 50 value of approximately 112 μM against both these cell lines, whereas a combination of NF and TAM required only half the concentrations of 50 μM and 10 μM to inhibit 50 % of the cell growth of each cell line. Since BRCA1, a tumor suppressor gene, has been implicated in the mechanism of tamoxifen induced growth inhibition, we determined the expression of this protein upon exposure of these cells to DIM. Results from western blot analysis show that there is a significant increase in BRCA1 and AhR protein levels in these cells upon treatment with DIM when compared to protein levels from untreated MCF-7 and MCF-7 /dox cells. Thus activation of BRCA1 by SAhRMs appears to be one of the mechanisms responsible for enhanced growth inhibitory effect of tamoxifen against these cells. These data suggest that either an AhR agonist or an antagonist can be conveniently employed in combination with TAM to increase the activity of TAM against both MCF-7 cells and doxorubicin resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5419.

In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence

We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect. We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3). In vitro, the growth of GL2-Dox, GL2+Dox, and R2-Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid beta-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2-Dox, GL2+Dox, and R2-Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation. In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.