Regulation Of Drug Sensitivity Research Articles

Targeting the IL-4/IL-13 signaling pathway sensitizes Hodgkin lymphoma cells to chemotherapeutic drugs

Hodgkin/Reed-Sternberg lymphoma (HL) is a clonal B-cell-related malignancy. Although many patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM-H2 as model systems, we investigated the role of IL-4/IL-13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL-4 and IL-13-mediated STAT6 activation by either an IL-4-binding fusion protein APG598 or an IL-4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5-Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6-mediated elevation of expression of the anti-apoptotic Bcl-2 family protein Bcl-xL. Employing ChIP analysis in combination with APG201 or STAT6-specific siRNA we identified a defined STAT6-binding site in the Bcl-xL promoter region from -1967 to -1957 of the transcription start site. Our data demonstrate that the IL-4/IL-13-STAT6-Bcl-xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL-4/IL-13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.

Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer

A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused by active drug efflux mediated by ATP-binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

The tumor microenvironment controls drug sensitivity

A better understanding of mechanisms involved in regulation of drug sensitivity is crucial for improved cancer treatment. New studies show that cells of the tumor microenvironment modulate the response of cancer cells to chemotherapy and targeted therapies through production of secreted factors ( pages 1359–1368 ).

Abstract 1534: Hes1 regulates hypoxia induced chemoresistance of myeloma cells

Abstract Bone marrow (BM) microenvironment plays a critical role in response of tumor cells to chemotherapeutic drugs. Hypoxia has been shown to contribute to pathogenesis not only solid but also hematological tumors. While normal BM environment is hypoxic, tumor growth is associated with increased hypoxia in BM niche. We have previously demonstrated that activation of Notch signaling could be one of the mechanisms responsible for survival of multiple myeloma (MM) cells from chemotherapeutic drugs. Here we investigated whether hypoxia could modulate Notch signaling and induce chemoresistance of MM cells. Three MM cell lines were treated with doxorubicin or melphalan in hypoxic (1% O2) or normoxic conditions. Using Annexin V binding assay we demonstrated that hypoxia induced chemoresistance in only one (H929) cell line studied. This effect was associated with significant up-regulation of expression of Notch target gene Hes1 and its protein level while Hes1 level was unchanged in other two MM cell lines. Inhibition of Hes1 with siRNA canceled the protective effect of hypoxia on MM cells. To further confirm the involvement of Notch pathway in regulation of drug sensitivity, Hes-1 was overexpressed in MM cells in normoxic condition using vector pIRES-AcGFP-Hes-1. MM cells with increased Hes1 level became less sensitive to chemotherapeutics as compared to cells transfected with control vector. Hes-1 is considered to be a Notch target gene. However, hypoxia did not affect Notch receptors expression level in MM cells. The effect of hypoxia on Hes1 was Notch-independent as was evident from Notch/CBF1 reporter assay. In addition, inhibition of Notch receptor cleavage with gamma-secretase inhibitor GSI-XII (Calbiochem) did not affect hypoxia induced MM cell chemoresistance. Up-regulation of Hes-1 was induced by HIF-1a as treatment of MM cells with hypoxia mimetic deferoxamine resulted in up-regulation of Hes1 and inhibition of HIF-1a with siRNA abrogated hypoxia-induced protection of MM cells. Thus, our study, for the first time, demonstrates that hypoxia could induce chemoresistance of MM cells through Notch-independent up-regulation of Hes-1. These results could be important not only for understanding the cancer biology but for designing therapeutic intervention in MM. 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 1534. doi:1538-7445.AM2012-1534

Abstract 3068: Hedgehog signaling regulates drug sensitivity by targeting ABC transporters in epithelial ovarian cancer (EOC)

Abstract A major challenge of successful chemotherapy in treatment of ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused mostly by the active efflux mediated by ATP-binding cassette (ABC) transporters. The regulation of these transporters in ovarian cancer is poorly understood. Our research shows that in ovarian cancer, abnormal regulation of the hedgehog (Hh) signaling pathway is involved in regulation of ABC transporters. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work on Gli1, a key Hh downstream signaling gene, has shown frequent activation of the Hh pathway in ovarian cancer. Inhibition of the Hh signaling pathway by inhibitors or Gli1 siRNA increases ABC gene expression levels and the response of ovarian cancer cells to certain chemotherapeutic drugs. The mechanism underlying is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABC genes. This study provides new information in the knowledge of ABC gene regulation and will contribute to generating new targets/markers to detect and anticipate ovarian cancer patient chemotherapy drug sensitivity which could lead to more effective treatments and improved survival. 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 3068. doi:1538-7445.AM2012-3068

Up-Regulation of Extracellular Signal-Regulated Kinase 1/2-Dependent Thymidylate Synthase and Thymidine Phosphorylase Contributes to Cisplatin Resistance in Human Non–Small-Cell Lung Cancer Cells

Chemotherapy for advanced human non-small-cell lung cancer (NSCLC) includes platinum-containing compound such as cisplatin in combination with a second- or third-generation cytotoxic agent. 5-Fluorouracil (5-FU) belongs to antimetabolite chemotherapeutics, and its mechanism of cytotoxicity is involved in the inhibition of thymidylate synthase (TS). TS and thymidine phosphorylase (TP) are key enzymes of the pyrimidine salvage pathway. In this study, we have examined the molecular mechanism of TS and TP in regulating drug sensitivity to cisplatin in NSCLC cell lines. Cisplatin could increase the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MKK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein levels of TS and TP through enhancing the protein stability in A549 and H1975 cells. Blocking ERK1/2 activation by MKK1/2 inhibitor [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene)] decreased TS and TP protein levels in both cell lines treated with cisplatin. Depletion of endogenous TS or TP expression by specific small interfering RNA transfection significantly increased cisplatin-induced cell death and growth inhibition. Combined treatment with 5-FU could decrease cisplatin-induced ERK1/2 activation and the induction of TS and TP, which subsequently resulted in synergistic cytotoxic effects. Enforced expression of constitutive active MKK1/2 vectors rescued the protein levels of phospho-ERK1/2, TS, and TP, and the cell viability that were decreased by cisplatin and 5-FU combination. In contrast, U0126 enhanced drug sensitivity to cisplatin and/or 5-FU in lung cancer cells. In conclusion, the up-regulation of ERK1/2-dependent TS and TP can protect human lung cancer cells from cisplatin-induced cytotoxicity.

MiR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and SW620 cell lines

Chemotherapy is an important treatment for colorectal adenocarcinoma cancer; however, colorectal adenocarcinoma cells often develop resistance to chemotherapeutic drugs, leading to relapse and poor patient prognosis. The development of drug resistance is often a multifactor process, which involved several genes and cellular mechanisms. microRNAs are endogenous small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the present study, we investigated the possible role of microRNAs in regulating drug sensitivity of colorectal adenocarcinoma cells SW620 and SW480. Using microRNA expression arrays and quantitative reverse transcriptase (RT)-PCR, we found that SW620 cells exhibited elevated miR-20a expression compared with SW480 cells. In addition, these two cell lines displayed different sensitivities to the chemotherapeutic drugs fluorouracil, oxaliplatin, and teniposide. Modulation of miR-20a altered the sensitivity of SW620 and SW480 cells to these drugs; knockdown of miR-20a sensitized SW620 cells to chemotherapeutic agents, whereas overexpression of miR-20a in SW480 cells resulted in chemoresistance. Endogenous BNIP2 mRNA and BNIP2 protein levels were inversely related to miR-20a levels as detected by quantitative RT-PCR and western blot analysis. Fluorescence reporter assays showed a direct interaction between miR-20a and the BNIP2 3'UTR. Taken together, our findings suggested that miR-20a may play a role in colorectal adenocarcinoma cancer cell drug resistance and may be a therapeutic target against chemotherapy drug resistance in colorectal adenocarcinoma.

Abstract 1035: Bcl2 is regulated by PKC and Akt and is associated with resistance to cisplatin in endometrial cancer cells.

Abstract Endometrial carcinomas often show resistance to cisplatin. Bcl2 has been shown to be associated with cisplatin resistance in different types of cancers. However, there is no information concerning its possible involvement in chemoresistance and its regulation in endometrial carcinomas. We have tested the ability of cisplatin to induce apoptosis in KLE, a resistant endometrial carcinoma cell line, and examined the putative role for Bcl2 in regulating drug sensitivity. We have investigated the impact of cisplatin treatment on Bcl2 family members and Xiap, an inhibitor of caspase activity. Cisplatin treatment upregulated Bcl2 protein expression in a time-dependant manner in KLE cells, but had no significant impact on the levels of Bax, Bcl-xL and Xiap proteins. As demonstrated by RT-PCR, Bcl2 mRNA was not upregulated in response to cisplatin, suggesting that cisplatin act on Bcl2 expression through a post-translational mechanism. In KLE cells, Bcl2 inhibition by HA14-1 led to increased apoptosis induced by cisplatin. It has been shown that Akt, MAPK and PKC pathways are involved in the regulation of Bcl2 under various conditions and in different cell types. Therefore, KLE cells were treated with inhibitors of MAPK (PD98059) and PKC (CalphostinC) prior to cisplatin treatment. Inhibition of PKC prevented cisplatin-dependant upregulation of Bcl2 protein, while MAPK inhibition had no impact. We previously showed that KLE cell line express high levels of phosphorylated Akt (pAkt) isoforms. Then, KLE cells stably expressing shRNA for all three Akt isoforms were established. Treatment of these cells with cisplatin prevented the increase of Bcl2 expression and led to increased apoptosis by cisplatin. These data suggest a role for Bcl2 in cisplatin resistance in endometrial carcinoma cells. Also, they indicate that PKC and Akt are involved in regulation of Bcl2 expression in this experimental model. 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 1035.

Expression and Role of hERG1 Channels in Pediatric Acute Lymphoblastic Leukemias: Shortcoming of Drug Resistance by hERG1 Channel Inhibitors in Stoma-Supported Leukaemia Cell Cultures In Vitro.

Therapy resistance is still a major obstacle to successful treatment in a significant number of pediatric acute lymphoblastic leukaemia (ALL) patients. It has been previously demonstrated that children with ALL whose leukemia cells exhibit in vitro resistance to single or a combination of drugs have a significantly worse prognosis compared to patients with sensitive leukemic cells (Ramakers-van Woerden N, et al Leukemia 18(3):521–9 2004). Ion channels are becoming one of the potential targets for cancer therapy and putative biochemical modulators of conventional chemotherapy (Conti M, J Exp Ther Oncol . 4(2):161–6, 2004). In particular, K+ channels belonging to the hERG1 family are attracting most attention, since they are over-expressed in a broad range of primary acute myeloid leukaemias (AML) as well as in both myeloid and lymphoid leukemic cell lines (Pillozzi S, et al Leukemia 16:1791–1798, 2002; Smith GA, et al, JBC 227:18528–18534, 2002). hERG1 channel expression confers a greater capacity to engraft the bone marrow and invade the bloodstream in NOD/SCID mice injected with AML cells. This fact corresponds to a greater malignancy (shorter overall survival and higher probability to relapse) in hERG1 positive AML patients (Pillozzi S, et al Blood 110:1238–1250, 2007). It was also recently shown that the expression of hERG1 is related to the chemosensitivity of cancer cells to vincristine, paclitaxel, and hydroxy-camptothecin (Chen SZ, et al Cancer Chemother Pharmacol 56(2):212–20, 2005). We studied the expression and role of hERG1 channels in various B lymphoid leukaemia cell lines and primary childhood B lymphoid leukaemia samples. It emerged that:i.hERG1 K+ channels are expressed in both all the leukaemia cell lines and primary childhood B leukaemia samples;ii.the N-terminus deleted, herg1b isoform was preferentially expressed in both cell lines and primary samples;iii.in childhood leukaemia patients, the level of herg1b expression correlated with response to therapy.B lymphoid leukaemia cell lines were co-cultured on human bone marrow stromal cells, a system known to enhance leukaemia cell survival and escape from drug-induced apoptosis. In these cultures, the addition of a specific hERG1 inhibitor, E4031, induced a significant apoptosis in leukaemia cells, bypassing the protective effect of the bone marrow microenvironment. We hypothesise that hERG1 channels can represent a novel molecular device regulating drug sensitivity in childhood acute leukaemia cells, and that targeting of hERG1 channels can restore a proper pro-apoptotic response to chemotherapy in resistant B lymphoid leukemic cells.

Circadian Rhythms, the Mesolimbic Dopaminergic Circuit, and Drug Addiction

Drug addiction is a devastating disease that affects millions of individuals worldwide. Through better understanding of the genetic variations that create a vulnerability for addiction and the molecular mechanisms that underlie the progression of addiction, better treatment options can be created for those that suffer from this condition. Recent studies point to a link between abnormal or disrupted circadian rhythms and the development of addiction. In addition, studies suggest a role for specific genes that make up the molecular clock in the regulation of drug sensitivity, sensitization, and reward. The influence of circadian genes and rhythms on drug-induced behaviors may be mediated through the mesolimbic dopaminergic system. This system has long been implicated in the development of addiction, and recent evidence supports a regulatory role for the brain's central pacemaker and circadian gene expression in the regulation of dopaminergic transmission. This review highlights the association between circadian genes and drug addiction, and the possible role of the mesolimbic dopaminergic system in this association.

Molecular analysis of mechanisms regulating drug sensitivity and the development of new chemotherapy strategies for genitourinary carcinomas.

The emergence of drug-resistant tumors during treatment remains one of the major obstacles in cancer chemotherapy. Overexpression of P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene or multidrug resistance-associated protein (MRP) (or both) and decreased expression of DNA topoisomerase II are responsible for expression of the multidrug resistance (MDR) phenotype. The expression of P-glycoprotein is also often observed in untreated cancers showing spontaneous MDR, such as renal cell carcinoma. Regarding cisplatin resistance, decreased cisplatin accumulation, an increase in cisplatin detoxification by glutathione-related enzymes or metallothionein (or both), and increased repair of DNA damage are all considered to play an important role. The combination of reversal agents targeting such drug resistance markers may be a way to improve the outcome of chemotherapy. Regarding the presently available reversal agents, however, clinically relevant chemosensitizing doses cannot be given to humans without inducing significant toxicity. The development of new agents that reverse drug resistance without causing significant toxicity and their clinical application based on the mechanisms regulating drug sensitivity may therefore be a potentially effective new treatment strategy for genitourinary carcinomas.

Erratum in Hu et al. Regulation of drug sensitivity by ribosomal protein S3a. Blood. 2000;95:1047-1055.

Erratum in Hu et al. Regulation of drug sensitivity by ribosomal protein S3a. Blood. 2000;95:1047-1055.

Putative P-Glycoprotein Expression in Arsenite-Resistant Leishmania donovani Down-Regulated by Verapamil

Western immunoblots of whole cell lysate and crude membrane extract of an in vitro selected sodium m-arsenite-resistant L. donovani strain revealed a 230-kDa protein identified by an anti-P-glycoprotein (Pgp) antibody. Immunofluorescence microscopy, using the same antibody, detected putative Pgp on resistant parasites. Overexpression of the putative Pgp was down-regulated by verapamil. These results provided, possibly, the first evidence that (i) overexpression of Pgp-like protein occurs in arsenite-resistant Leishmania that are cross-resistant to structurally and functionally unrelated drugs and (ii) verapamil regulates drug sensitivity possibly by down-regulating Pgp expression in the arsenite-resistant Leishmania.

Regulation of drug sensitivity by ribosomal protein S3a

When bcl-2 is immunoprecipitated from (32)P-labeled cell extracts of all-trans retinoic acid (ATRA)-treated acute myeloblastic leukemia (AML) blasts, a phosphorylated protein of approximately 30 kd is coprecipitated. This protein has been identified as ribosomal protein S3a. The biologic effects of S3a include favoring apoptosis and enhancing the malignant phenotype. We sought to determine whether S3a, like bcl-2, influenced the response of cells to chemotherapeutic drugs and ATRA. Cell lines were studied in which S3a was genetically increased or disrupted; increased S3a was regularly associated with increased plating efficiency and increased sensitivity to either cytosine arabinoside (ara-C) or doxorubicin (DNR). S3a did not affect the sensitivity of cells to paclitaxel. Pulse exposures to either (3)HTdR or ara-C showed a greater percentage of clonogenic cells in the S phase of the cell cycle in cells with increased S3a than in controls. Cells with increased S3a responded to ATRA by increased ara-C or DNR sensitivity, whereas cells with reduced S3a protein were either protected by ATRA or not affected. We studied cryopreserved blast cells from patients with AML or chronic myelomonocytic leukemia (CMML). S3a protein levels were heterogeneous in these populations. In 32 cryopreserved blast populations, S3a levels were significantly correlated with both bcl-2 and with cell growth in culture. As in cell lines, high S3a in cryopreserved blasts was associated with ATRA-induced sensitization to ara-C. No significant association was seen between S3a levels and response to treatment.