Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.     

Phosphorylation of RNA helicase A by DNA-dependent protein kinase is indispensable for expression of the MDR1 gene product P-glycoprotein in multidrug-resistant human leukemia cells

Development of multidrug resistance (MDR) in cancer frequently involves overexpression of the MDR1 gene product P-glycoprotein (P-gp), a drug transporter which severely impedes the efficacy of chemotherapy. Because intensive efforts to identify therapeutics that reverse MDR by inhibiting the drug transport activity of P-gp have not yet met with success, we have focused on the alternative strategy of targeting MDR1 promoter activation to knockdown P-gp expression in cancer cells. We recently identified RNA helicase A (RHA) inhibition as a rational strategy to downregulate P-gp in leukemia cells by showing that RHA RNAi knockdown abrogated P-gp expression in MDR variants of human leukemia HL-60 cells. In that report, we also demonstrated that RHA activated the MDR1 promoter in the MDR variant cells but not in the drug-sensitive counterpart. This led us to hypothesize that P-gp induction by RHA required cooperation with another factor present only in the MDR variants. Here, we identify the RHA cooperating factor as DNA-PK catalytic subunit (cs), and we show that DNA-PKcs resides with RHA at the MDR1 promoter in a multiprotein complex. Furthermore, targeted DNA-PKcs inhibition abrogated P-gp expression in the MDR variant cells. We demonstrate that constitutive multisite RHA phosphorylation producing retarded migration in SDS-PAGE is catalyzed by DNA-PKcs in the MDR variants, and does not occur in the parental cells, which are DNA-PKcs deficient. The indispensable role played by DNA-PK in P-gp overexpression in MDR leukemia cells in this report identifies targeted DNA-PK inhibition as a rational strategy to reverse drug resistance in cancer.     

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.     

长春新碱诱导建立人结肠癌多药耐受性小鼠模型及MDR1和MRP1基因表达

目的建立人结肠癌多药耐受性动物模型并初步探索其耐药机制。方法结合体内外诱导方法建立人结肠癌多药耐受性动物模型,利用VCR和CTX的肿瘤抑制实验评价其MDR特性;利用real-time PCR和West-ern blotting等方法分析其P-gp/MDR1和MRP1基因和蛋白的表达。结果肿瘤抑制实验结果显示,MDR和敏感型结肠癌模型的肿瘤生长速度差异不显著,MDR结肠癌动物模型对于VCR和CTX的耐药性均有较大程度的提高;表达分析结果显示,人结肠癌MDR动物模型的P-gp/MDR1表达水平有较大提高,而MRP1表达没有显著变化。结论人结肠癌多药耐受性动物模型具有较好的多药耐受性,其多药耐受性表型主要是由于P-gp/MDR1过量表达所导致。     

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza

ObjectiveMultidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine.MethodsThe inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones.ResultsBi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity.ConclusionsTaken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.     

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance

Despite the notable efficacy of oxaliplatin in the treatment of colorectal cancers, the metastatic tumours ultimately become resistant to the drug. This study investigated whether the oxaliplatin-resistant cells display different behaviour to this drug versus the sensitive cells and if this difference may be further exploited into the clinical treatments improvement. In order to establish a stable cell line resistant to oxaliplatin, a human colorectal cancer cell line (Colo320) was exposed to increasing doses of the drug up to the clinically relevant plasma concentration. Four cell groups with different levels of chemoresistance were subjected to additional doses of oxaliplatin, and their cytotoxicity, apoptosis and DNA damage production were assessed. Cells selected for resistance to oxaliplatin reacted differently to the application of additional doses of the drug, displaying lower toxicity and cellular death and fewer DNA cross-links formation, in accordance with the extent of the oxaliplatin pretreatments. As the cross-links formation by oxaliplatin being the main cause for cytotoxicity of this drug and a correlation between cytotoxicity and clinical outcome being shown repeatedly, we consider that the evaluation of oxaliplatin-induced cytotoxicity, apoptosis and DNA damage could be a valuable tool to assess the tumour cells sensitivity and thus to predict the response to chemotherapy.     

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background

The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells.

Principal Findings

For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53^+/+, RHCT116 p53^−/−) from the parental HT29, HCT116 p53^+/+ and HCT116 p53^−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53^−/− but not in the RHCT116 p53^+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53^−/− but not in the RHCT116 p53^+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells.

Conclusions

Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.     

<Emphasis Type="Italic">In Silico</Emphasis> Quantitative Structure-Activity Relationship Studies on P-gp Modulators of Tetrahydroisoquinoline-Ethyl-Phenylamine Series

Background  

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The drug efflux by a transport protein is the main reason for MDR. In humans, MDR mainly occurs when the ATP-binding cassette (ABC) family of proteins is overexpressed simultaneously. P-glycoprotein (P-gp) is most commonly associated with human MDR; it utilizes energy from adenosine triphosphate (ATP) to transport a number of substrates out of cells against concentration gradients. By the active transport of substrates against concentration gradients, intracellular concentrations of substrates are decreased. This leads to the cause of failure in cancer chemotherapy.     

Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin,Paclitaxel, Doxorubicin,and Fluorouracil in Part via microRNA-107

Higher Lin28 expression is associated with worse pathologic tumor responses in locally advanced gastric cancer patients undergoing neoadjuvant chemotherapy. However, the characteristics of Lin28 and its mechanism of action in chemotherapy resistance is still unclear. In this study, we found that transfection of Lin28 into gastric cancer cells (MKN45 and MKN28) increased their resistance to the chemo-drugs oxaliplatin (OXA), paclitaxel (PTX), doxorubicin (ADM), and fluorouracil (5-Fu) compared with gastric cancer cells transfected with a control vector. When knockdown Lin28 expression by Lin28 small interfering RNA (siRNA) was evaluated in vitro, we found that the resistance to chemo-drugs was reduced. Furthermore, we found that Lin28 up-regulates C-myc and P-gp and down-regulates Cylin D1. Finally, we found that miR-107 is a target microRNA of Lin28 and that it participates in the mechanism of chemotherapy resistance. Our results suggest that the Lin28/miR-107 pathway could be one of many signaling pathways regulated by Lin28 and associated with gastric cancer chemo-resistance.     

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells

Multi-drug resistance (MDR), an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC) transporters and activated Sonic hedgehog (Shh) signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX), we examined the effect and mechanism of norcantharidin (NCTD), a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S) and DOX-resistant (MCF-7R) cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.     

Gene network analysis of oxaliplatin-resistant colorectal cancer to target a crucial gene using chitosan/hyaluronic acid/protamine polyplexes containing CRISPR-Cas9

Colorectal cancer (CRC) treatment is dramatically hampered by resistance to oxaliplatin alone or in the combination of irinotecan or 5-fluorouracil and leucovorin. This study aims to design and assess Chitosan/Hyaluronic Acid/Protamine sulfate (CS/HA/PS) polyplexes loaded with CRISPR plasmid for targeting a key gene in cancer drug resistance. Here, recent findings were considered to validate oxaliplatin-resistant CRC-related genes and systems biology approaches employed to detect the critical gene. The polyplexes were characterized according to particle size, zeta potential, and stability. Moreover, carrier toxicity and transfection efficiency were assessed on oxaliplatin-resistant HT-29 cells. The post-transfection evaluations were performed to confirm gene disruption-mediated CRISPR. Eventually, excision cross complementation group 1(ERCC1), a crucial member of the nucleotide excision repair pathway, was selected to be targeted using CRISPR/Cas9 to reverse oxaliplatin resistance in HT-29 cells. CS/HA/PS polyplexes containing CRISPR/Cas9 plasmid exhibited negligible toxicity and comparable transfection efficiency with Lipofectamine™. Following the efficient gene delivery, sequences in CRISPR/Cas9 target sites were altered, ERCC1 was downregulated, and drug sensitivity was successfully restored in oxaliplatin-resistant cells. Findings indicate that CS/HA/PS/CRISPR polyplexes provide a potential strategy for delivering cargo and targeting oxaliplatin resistance-related gene to manipulate drug resistance as a rising concern in cancer therapeutic approaches.