Multiple myeloma (MM) is the second most common blood cancer. Treatments for MM include corticosteroids, alkylating agents, anthracyclines, proteasome inhibitors, immunomodulatory drugs, histone ...deacetylase inhibitors and monoclonal antibodies. Survival outcomes have improved substantially due to the introduction of many of these drugs allied with their rational use. Nonetheless, MM patients successively relapse after one or more treatment regimens or become refractory, mostly due to drug resistance. This review focuses on the main drugs used in MM treatment and on causes of drug resistance, including cytogenetic, genetic and epigenetic alterations, abnormal drug transport and metabolism, dysregulation of apoptosis, autophagy activation and other intracellular signaling pathways, the presence of cancer stem cells, and the tumor microenvironment. Furthermore, we highlight the areas that need to be further clarified in an attempt to identify novel therapeutic targets to counteract drug resistance in MM patients.
Overexpression of P‐glycoprotein (P‐gp) contributes to the multidrug resistance (MDR) phenotype found in many cancer cells. P‐gp has been identified as a promising molecular target, although attempts ...to find successful therapies to counteract its function as a drug efflux pump have largely failed to date. Apart from its role in drug efflux, P‐gp may have other cellular functions such as being involved in apoptosis, and is found in various locations in the cell. Its expression is highly regulated, namely by microRNAs (miRNAs or miRs). In addition, P‐gp may regulate the expression of miRs in the cell. Furthermore, both P‐gp and miRs may be found in microvesicles or exosomes and may be transported to neighboring, drug‐sensitive cells. Here, we review this current issue together with recent evidence of this network of interactions between P‐gp and miRs.
Overexpression of oncomiR-21 has been observed in most cancer types, such as leukemia. This miR has been implicated in a number of cellular processes, including chemoresistance, possibly by directly ...modulating the expression of several apoptotic related proteins. It was recently shown to directly target Bcl-2 mRNA and upregulate Bcl-2 protein expression. Nevertheless, the possible effect of miR-21 in autophagy has never been addressed. This study investigates the effects of targeting miR-21 with antimiRs on chronic myeloid leukemia cellular autophagy and on associated drug sensitivity. We observed that miR-21 downregulation decreased cellular viability and proliferation, although no changes to the normal cell cycle profile were observed. miR-21 downregulation also caused increased programmed cell death and a decrease in the expression levels of Bcl-2 protein, although PARP cleavage was not affected, indicating that apoptosis was not the relevant mechanism underlying the observed results. Treatment with antimiR-21 caused an increase in the autophagy related proteins Beclin-1, Vps34 and LC3-II. Accordingly, autophagic vacuoles were visualized both by monodansylcadaverine (MDC) and acridine orange (AO) staining and also by transmission electron microscopy (TEM). Additionally, miR-21 downregulation increased K562 and KYO-1 cellular sensitivity to etoposide or doxorubicin. This chemosensitivity was reverted by pre-treating cells with 3-MA, an autophagy inhibitor. Finally, serum starvation (an autophagy inducer) also increased sensitivity to these drugs, confirming that autophagy sensitized these cells to the effect of these drugs. To the best of our knowledge, this is the first description of autophagy induction via miR-21 targeting and its involvement in drug sensitivity.
The virtual screening of a library of chalcone derivatives led us to the identification of potential new MDM2 ligands. The chalcones with the best docking scores obeying the Lipinski rule of five ...were subsequently prepared by base-catalyzed aldol reactions. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast-based screening assay. Using this approach two chalcones (3 and 4) were identified as putative small molecule inhibitors of p53–MDM2 interaction. The activity of both chalcones was further investigated in a panel of human tumor cells. Chalcones 3 and 4 revealed a pronounced tumor cell growth inhibitory effect on tumor cell lines. Additionally, chalcone 4 caused alterations in the cell cycle profile, induced apoptosis and increased the levels of p53, p21 and PUMA proteins in NCI-H460 cells. Computational docking studies allowed to predict that, like nutlin-3A (a well-known small-molecule inhibitor of p53–MDM2 interaction), chalcones 3 and 4 bind to the p53-binding site of MDM2. The results here presented will be valuable for the structure-based design of novel and potent p53–MDM2 inhibitors.
Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell ...growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1·HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1·HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1·HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis.
miR-128 has been associated with cancer, particularly with leukemia. In particular, this miR has been described, together with other miRs, to allow the discrimination between AML (acute myeloid ...leukemia) and ALL (acute lymphoblastic leukemia). In addition, miR-128 is included in miR signatures which not only allow characterizing a particular subtype of AML but are also associated with worse clinical outcome in a subgroup of patients with high-risk molecular features of AML. Nevertheless, all the published studies are based on data from expression arrays and no functional studies have been performed. Therefore, in order to further understand the role of miR-128 in AML cells and in their response to some chemotherapy, overexpression of miR-128 was achieved with miR-mimics in an AML cell line (HL-60). This resulted in decreased cellular viability and increased sensitization to both etoposide and doxorubicin. Overexpression of miR-128 increased programmed cell death but had no effect on cell cycle profile, 1 apoptosis or autophagy, as no alterations were observed in the protein levels of PARP, pro-caspase-3, Vps34, Beclin-1 or LC3-II. In addition, miR-128 overexpression increased the levels of DNA damage, as could be concluded by an increase in the comet's tail intensity in the comet assay, an increase in the number of DNA repair foci stained with either γ-H2AX or 53BP1 proteins, and an increase in the levels of these two proteins (observed by Western blot). To the best of our knowledge, this is the first association of miR-128 with DNA damage in a leukemia context.
Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF ...pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.
Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.
Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF(V600E) mutation. In BRAF(V600E) mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27(Kip1). Specific inhibition of BRAF by RNAi in cells with BRAF(V600E) mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF(V600E) mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.
Our results in thyroid cancer cells, namely those harbouring BRAF(V600E) mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF(V600E) mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.
To date, there are no simple and minimally invasive methods to diagnose MDR. Extracellular vesicles (EVs) are shed by all cells, carry a specific cargo from the donor cells and are present in several ...body fluids, which means that they can potentially be easily collected from cancer patients and become the source of biomarkers to diagnose cancer. This data article contains a full list of the proteins identified in the EVs shed by an isogenic pair of chronic myeloid leukaemia cells (MDR cells and their drug-sensitive counterparts) by LC/MS/MS analysis, together with their GeneOntology analysis. In addition, it also contains data from protein content analysis and Dynamic light scattering count-rate events of the referred EVs as well as of the EVs shed from an isogenic pair of non-small cell lung cancer cells (MDR cells and their drug-sensitive counterparts). The interpretation of the data presented in this article and further extensive insights can be found in “Multidrug resistant tumour cells shed more microvesicles-like EVs and less exosomes than their drug-sensitive counterpart cells” 1.
Multidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major ...clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.
Two pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.
We found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.
The determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.
This work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.
•MDR cells release bigger EVs than their drug-sensitive counterparts.•EVs released by MDR cells have less proteins involved in exosome biogenesis.•MDR cells shed more microvesicle-like EVs than exosomes.•Raises the possibility of developing a minimally-invasive method of detecting MDR
Abstract
P-glycoprotein (P-gp) overexpression is a major cause of multidrug resistance (MDR) and presents a serious challenge to the efficient treatment of cancer1. In spite of being a major clinical ...challenge, to date there are no non-invasive means to diagnose the MDR phenotype, using non-tumour biological samples. Recently, the non-genetic acquisition of functional P-gp has been described as being possibly mediated by extracellular vesicles (EVs). Indeed, P-gp may be transferred by EVs from MDR cells to drug-sensitive (DS) recipient cells3. Collectively, EVs may refer to exosomes (with endosomal origin and sizes ranging from 30-100nm), microvesicles (with plasma membrane origin and sizes ranging from 50-2000nm) or even apoptotic bodies (ranging from 50-5000nm)4.
This study aimed at characterizing and comparing EVs from P-gp-overexpressing MDR cancer cells and their respective DS counterparts, as an attempt to identify MDR biomarkers.
Two pairs of MDR cancer cell lines and their DS counterparts were used: a chronic myeloid leukaemia pair (K562Dox, a kind gift from Dr. Jean-Pierre Marie and K562, from EACC) and a non-small cell lung cancer pair (RH460 and NCI-H460, a kind gift from Dr. Milica Pesic). EVs shed by these cells were characterized in terms of size and molecular markers. In addition, their protein content was investigated by proteomic analysis and by Western blot.
This study describes, for the first time, that P-gp overexpressing MDR cells produce larger EVs than their DS counterparts. In addition, a distinct protein signature was found between the EVs shed by DS cells and their MDR counterparts, consisting mainly in differences in the relative abundances of particular proteins. As predicted, P-gp was found present only in the EVs shed by MDR cells. Interestingly, proteins involved in the biogenesis of exosomes, such as Syntenin-1, TSG-101, CD63, Clathrin LCB and CHMP4, were found decreased in EVs shed by MDR cells. These results were in agreement with the identified size differences between the EVs from MDR and DS cells mentioned above, which may suggest that MDR cells produce more microvesicles and that DS cells produce more exosomes.
In conclusion, it is proposed that EVs shed by the P-gp overexpressing MDR cells may have a specific size and protein signature, consisting in P-gp presence and a lower abundance of proteins involved in the biogenesis of exosomes. This may allow the development of a non-invasive way of detecting and predicting MDR, by analysing EVs shed into the circulation of cancer patients.
1. Lopes-Rodrigues, V. et al. The network of P-glycoprotein and microRNAs interactions. Int J Cancer 135, 253-263, (2014).
2. Bebawy, M. et al. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells. Leukemia 23, 1643-1649, (2009).
3. Akers, J. C. et al. Biogenesis of extracellular vesicles (EV). J Neurooncol 113, 1-11, (2013).
Citation Format: Vanessa Lopes-Rodrigues, Alessio Di Luca, Diana Sousa, Hugo Seca, Paula Meleady, Michael Henry, Raquel T. Lima, Robert O'Connor, M. Helena Vasconcelos. P-gp-overexpressing MDR cells shed larger extracellular vesicles than their drug-sensitive counterparts. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2015-4433
PDF
