Preferential Energy- and Potential-Dependent Accumulation of Cisplatin–Gutathione Complexes in Human Cancer Cell Lines (GLC4 and K562): A Likely Role of Mitochondria

cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20–30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.     

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma.Subject terms: Cancer therapeutic resistance, Mitophagy     

Reversal of MRP-mediated multidrug resistance in human lung cancer cells by the antiprogestatin drug RU486.

Multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) are drug efflux pumps conferring multidrug resistance to tumor cells. RU486, an antiprogestatin drug known to inhibit P-gp function, was examined for its effect on MRP activity in MRP-overexpressing lung tumor GLC4/Sb30 cells. In such cells, the antihormone compound was found to increase intracellular accumulation of calcein, a fluorescent compound transported by MRP, in a dose-dependent manner, through inhibition of cellular export of the dye; in contrast, it did not alter calcein levels in parental GLC4 cells. RU486, when used at 10 microM, a concentration close to plasma concentrations achievable in humans, strongly enhanced the sensitivity of GLC4/Sb30 cells towards two known cytotoxic substrates of MRP, the anticancer drug vincristine and the heavy metal salt potassium antimonyl tartrate. Vincristine accumulation levels were moreover up-regulated in RU486-treated GLC4/Sb30 cells. In addition, such cells were demonstrated to display reduced cellular levels of glutathione which is required for MRP-mediated transport of some anticancer drugs. These findings therefore demonstrate that RU486 can down-modulate MRP-mediated drug resistance, in addition to that linked to P-gp, through inhibition of MRP function.     

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-methyl-pyrazolate)]²⁺ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na⁺/K⁺-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.     

Impaired hydrolysis of cisplatin derivatives to aquated species prevents energy-dependent uptake in GLC4 cells resistant to cisplatin

It has been widely stated that cisplatin enters cells by passive diffusion, despite some reports supporting a carrier-mediated mechanism. We have determined the rate of uptake of carboplatin (CBDCA), of cisplatin (CDDP) and of aquated forms, at different values of the extracellular pH, in the small lung-cancer cells GLC4 and GLC4/CDDP, cells resistant to CDDP. The rate of CDDP uptake is about 2-fold lower in resistant cells than in sensitive ones; in ATP-depleted cells this rate is about the same for both cell lines. The rate of CBDCA uptake is about 10-fold lower than that of CDDP and is the same in both cell lines independently of the ATP status of the cells. On the other hand, the rate of uptake of the aquated form of CDDP is approximately 40-fold higher than that of CDDP and is the same in both cell lines, but decreases dramatically in ATP-depleted cells. The plot of the initial rate of uptake of the aquated species as a function of its extracellular concentration shows a tendency to be saturable with k(m)=1.9 mM. In conclusion, our data show that, in sensitive GLC4 cells, passive diffusion of CDDP, probably in its neutral dichloro form, and active uptake of the aquated form contribute to the platinum uptake. The active transport of CDDP involves at least two steps: (1). the hydrolysis of the dichloro species in a deficient Cl(-) space at the level of the plasma membrane, which is the limiting step, and (2). the active transport of the aquated species. In resistant cells, step (1). should be deficient whereas step (2). is the same as in sensitive cells. For CBDCA this mechanism holds; however, step (1). is so low that the active transport does not contribute to the uptake of CBDCA by cells.     

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.     

The MRP1-mediated effluxes of arsenic and antimony do not require arsenic-glutathione and antimony-glutathione complex formation

Arsenic trioxide is an effective treatment for acute promyelocytic leukemia, but resistance to metalloïd salts is found in humans. Using atomic absorption spectroscopy, we have measured the rate of uptake of arsenic trioxide and of antimony tartrate in GLC4 and GLC4/ADR cells overexpressing MRP1 and the rate of their MRP1-mediated effluxes as a function of the intracellular GSH concentration. In sensitive cells, after 1 h, a pseudosteady state is reached where intra- and extracellular concentrations of metalloid are the same. This precludes the formation, at short term, of complexes between arsenic or antimony with GSH. In resistant cells reduced intracellular accumulation of arsenic (or antimony), reflecting an increased rate of arsenic (or antimony) efflux from the cells, is observed. No efflux of the metalloid is observed in GSH depleted cells. The two metalloïds and GSH are pumped out by MRP1 with the same efficiency. Moreover for the three compounds 50% of the efflux is inhibited by 2 M MK571. This led us to suggest that As- and Sb-containing species could be cotransported with GSH.     

Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor. Although cisplatin is the primary chemotherapy used in osteosarcoma treatment, the cisplatin resistance remains a big challenge for improving overall survival. The store-operated calcium (Ca²⁺) entry (SOCE) and its major mediator Stim1 have been shown to be implicated in a number of pathological processes typical for cancer. In this study, we showed that Stim1 expression was significantly increased in chemo-resistant osteosarcoma tissues compared with chemo-sensitivity tissues. Patients with Sitm1 expression exhibited poorer overall survival than Stim1-negative patients. Moreover, un-regulation of Stim1 expression and SOCE were also observed in cisplatin-resistant MG63/CDDP cells compared with their parental cells. Cisplatin treatment obviously reduced Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, cisplatin resulted in a more pronounced increase of endoplasmic reticulum (ER) stress in MG63 cells than in their resistant variants, which was evidenced by the activation of molecular markers of ER stress, GRP78, CHOP and ATF4. Knockdown of Stim1 using siRNA remarkably enhanced cisplatin-induced apoptosis and ER stress in MG63/CDDP cells, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of Stim1 markedly reversed apoptosis and ER stress following cisplatin treatment. Taken together, these results demonstrate that Stim1 as well as Ca²⁺ entry contributes cisplatin resistance via inhibition of ER stress-mediated apoptosis, and provide important clues to the mechanisms involved in cisplatin resistance for osteosarcoma treatment. Stim1 represents as a target of cisplatin and blockade of Stim1-mediated Ca²⁺ entry may be a useful strategy to improve the efficacy of cisplatin to treat osteosarcoma.     

Stabilization of p73 by nuclear IkappaB kinase-alpha mediates cisplatin-induced apoptosis

In response to DNA damage, p53 and its homolog p73 have a function antagonistic to NF-kappaB in deciding cell fate. Here, we show for the first time that p73, but not p53, is stabilized by physical interaction with nuclear IkappaB kinase (IKK)-alpha to enhance cisplatin (CDDP)-induced apoptosis. CDDP caused a significant increase in the amounts of nuclear IKK-alpha and p73alpha in human osteosarcoma-derived U2OS cells. Ectopic expression of IKK-alpha prolonged the half-life of p73 by inhibiting its ubiquitination and thereby enhancing its transactivation and pro-apoptotic activities. Consistent with these results, small interfering RNA-mediated knockdown of endogenous IKK-alpha inhibited the CDDP-mediated accumulation of p73alpha. The kinase-deficient mutant form of IKK-alpha interacted with p73alpha, but failed to stabilize it. Furthermore, CDDP-mediated accumulation of endogenous p73alpha was not detected in mouse embryonic fibroblasts (MEFs) prepared from IKK-alpha-deficient mice, and CDDP sensitivity was significantly decreased in IKK-alpha-deficient MEFs compared with wild-type MEFs. Thus, our results strongly suggest that the nuclear IKK-alpha-mediated accumulation of p73alpha is one of the novel molecular mechanisms to induce apoptotic cell death in response to CDDP, which may be particularly important in killing tumor cells with p53 mutation.     

High-Level Resistance to Cobalt and Nickel but Probably No Transenvelope Efflux: Metal Resistance in the Cuban Serratia marcescens Strain C-1

Molecular mechanisms underlying inducible cobalt and nickel resistance of a bacterial strain isolated from a Cuban serpentine deposit were investigated. This strain C-1 was assigned to Serratia marcescens by 16S rDNA analysis and DNA/DNA hybridization. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for cobalt- and nickel-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The predicted NcrA product was a NreB ortholog of the major facilitator protein superfamily and central for cobalt/nickel resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in Escherichia coli and caused decreased accumulation of Co(II) and Ni(II) in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the nickel–cobalt transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni(II) and Co(II). Because the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.     

Translational regulation of the mRNA encoding the ubiquitin peptidase USP1 involved in the DNA damage response as a determinant of Cisplatin resistance

Cisplatin (cis-diaminedichloroplatin (II), CDDP) is part of the standard therapy for a number of solid tumors including Non-Small-Cell Lung Cancer (NSCLC). The initial response observed is in most cases only transient and tumors quickly become refractory to the drug. Tumor cell resistance to CDDP relies on multiple mechanisms, some of which still remain unknown. In search for such mechanisms, we examined the impact of CDDP on mRNA translation in a sensitive and in a matched resistant NSCLC cell line. We identified a set of genes whose mRNAs are differentially translated in CDDP resistant vs. sensitive cells. The translation of the mRNA encoding the Ubiquitin-Specific Peptidase 1 (USP1), a Ubiquitin peptidase with important function in multiple DNA repair pathways, is inhibited by CDDP exposure in the sensitive cells, but not in the resistant cells. This lack of down-regulation of USP1 expression at the translational level plays a primary role in CDDP resistance since inhibition of USP1 expression or activity by siRNA or the small molecule inhibitor ML323, respectively is sufficient to re-sensitize resistant cells to CDDP. We involved the USP1 mRNA translation as a major mechanism of CDDP resistance in NSCLC cells and suggest that USP1 could be evaluated as a candidate predictive marker and as a therapeutic target to overcome CDDP resistance. More generally, our results indicate that analysis of gene expression at the level of mRNA translation is a useful approach to identify new determinants of CDDP resistance.     

OLFM4 depletion sensitizes gallbladder cancer cells to cisplatin through the ARL6IP1/caspase-3 axis

BackgroundGallbladder cancer (GBC) is a highly lethal malignancy that carries an extremely poor prognosis due to its chemoresistant nature. Cisplatin (CDDP) is a first-line chemotherapeutic for GBC; however, patients experienced no benefit when treated with CDDP alone. The underlying mechanisms of CDDP resistance in GBC remain largely unknown.MethodsAgilent mRNA microarray analysis was performed between paired GBC and paracarcinoma to explore differentially expressed genes that might underlie drug resistance. Gene Set Enrichment Analysis (GSEA) was employed to identify key genes mediating CDDP resistance in GBC, and immunohistochemistry was performed to validate protein expression and test correlations with clinicopathological features. In vitro and in vivo functional assays were performed to investigate the proteins’ roles in CDDP resistance.ResultsOlfactomedin 4 (OLFM4) was differentially expressed between GBC and paracarcinoma and had the highest rank metric score in the GSEA. OLFM4 expression was increasingly upregulated from chronic cholecystitis to GBC in clinical tissue samples, and OLFM4 depletion decreased GBC cell proliferation and invasion. Interestingly, downregulation of OLFM4 reduced ARL6IP1 (antiapoptotic factor) expression and sensitized GBC cells to CDDP both in vitro and in vivo. The evidence indicated that CDDP could significantly increase Bax and Bad expression and activate caspase-3 cascade in OLFM4-depleted GBC cells through ARL6IP1. Clinically, lower OLFM4 expression was associated with good prognosis of GBC patients.ConclusionsOur results suggest that OLFM4 is an essential gene that contributes to GBC chemoresistance and could serve as a prognostic biomarker for GBC. Importantly, OLFM4 could be a potential chemotherapeutic target.     

Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II)

A series of murine thymic lymphoma cell sublines was selected in vitro for resistance to cis-diamminedichloroplatinum(II) (CDDP). The level of CDDP resistance correlated with reduced drug accumulation in these cells. A rabbit antiserum was raised against the plasma membrane of a CDDP-resistant subline and used in Western blot analyses. Increased expression of a surface antigen of approximately 200 kDa was observed and found to correlate with the degree of resistance. Further biochemical and immunological studies demonstrated that this is a plasma membrane glycoprotein. However, it is different from the multidrug resistance-associated P-glycoprotein with a molecular weight of about 170,000. We have called this unique CDDP resistance-associated membrane protein CPR-200.     

Mitochondrial comparative proteomics of human ovarian cancer cells and their platinum-resistant sublines

Resistance to platinum-based chemotherapy is the major obstacle to successful treatment of ovarian cancer. It is evident that mitochondrial defects and the dysfunctions of oxidative phosphorylation and energy production in ovarian cancer cells were directly related to their resistance to platinum drugs. Using 2-D DIGE, we compared mitochondrial proteins from two platinum-sensitive human ovarian cancer cell lines (SKOV3 and A2780) with that of four platinum-resistant sublines (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP). Among the 236 differentially expressed spots, five mitochondrial proteins (ATP-α, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain were identified through mass spectrometry. All of them are downregulated in one or two of the platinum-resistant cell lines. Three proteins (ATP-α, PRDX3, and PHB) were validated by using western blot and immunohistochemistry. There is a significant decrease of PHB in tumor tissues from ovarian cancer patients who were resistant to platinum-based chemotherapies. This is the first direct mitochondrial proteomic comparison between platinum-sensitive and resistant ovarian cancer cells. These studies demonstrated that 2-D DIGE-based proteomic analysis could be a powerful tool to investigate limited mitochondrial proteins, and the association of PHB expression with platinum resistance indicates that mitochondria defects may contribute to platinum resistance in ovarian cancer cells.     

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II).

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.     

Intracellular L-arginine concentration does not determine NO production in endothelial cells: implications on the "L-arginine paradox"

We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of (15)N(4)-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, (15)N(4)-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by(15)N-nitrite or estimated (15)N(3)-citrulline concentrations when (15)N(4)-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced (15)N(4)-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by (15)N-nitrite, total nitrite and (15)N(3)-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the "L-arginine paradox" should not consider intracellular ARG concentration as a reference point.     

Oil body formation in Euphorbia tirucalli L. cell suspension cultures

The culture of cells of Euphorbia tirucalli L. resulted in the formation of intracellular particles which osmium tetroxide staining and electron microscopy indicated to be oil bodies. Chemical analysis (TLC, HPLC, GLC and GC-MS) disclosed steroids, triterpenoids, and diterpenoids as the major oil components of the cultured cells.