Intracellular Platinum Concentration Research Articles

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion.

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.

Ultrasmall iron oxide nanoparticles cisplatin (IV) prodrug nanoconjugate: ICP-MS based strategies to evaluate the formation and drug delivery capabilities in single cells

Ultrasmall iron oxide nanoparticles (<10 nm) were explored here as nanotransporters of cis-diamminetetrachloroplatinum (IV) (a cisplatin prodrug) in cellular models. The coating of the particles containing reactive carboxylic acid groups enabled the formation of a stable conjugate between the prodrug and the nanoparticles using one pot reaction. The nanoconjugate was characterized by different techniques exhibiting diameters of about 6.6 ± 1.0 nm. The use of a hyphenated strategy based on high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) permitted the quantitative evaluation of Fe and Pt in the nanoconjugate. Furthermore, the cellular uptake of the synthetic nanoconjugate was explored by single cell-ICP-MS (SC-ICP) which was used for the first time in this type of studies. The experiments in A2780 and A2780cis, sensitive and resistant ovarian cancer cell models respectively, revealed intracellular platinum concentrations of 12 fg/cell and 4 fg/cell, respectively which were 4-fold higher with respect to the uptake of cisplatin in both models. Intracellular drug release from the nanoconjugate was proved by measuring DNA platination in the same cells. In this case, levels of about 250 ng Pt/mg DNA were observed, about 5-fold higher when the nanoconjugate was used in comparison to cisplatin. Furthermore, the differences between the two lines turned to be significantly smaller than in the case of using cisplatin. The quantitative analytical tools developed here provided essential information required to fully characterize the developed nanoplatforms particularly important to overcome drug resistance.

Nanotechnology-Based Cisplatin Intracellular Delivery to Enhance Chemo-Sensitivity of Ovarian Cancer.

BackgroundPlatinum resistance is a major challenge in the management of ovarian cancer. Even low levels of acquired resistance at the cellular level lead to impaired response to cisplatin. In ovarian cancer intraperitoneal therapy, nanoparticle formulation can improve the cisplatin’s pharmacokinetics and safety profile.PurposeThis work aimed to investigate the chemo-sensitivity of ovarian cancer SKOV3 cells upon short-term (72h) single treatment of cisplatin and cisplatin-loaded biodegradable nanoparticles (Cis-NP). The aim was then to determine the therapeutic properties of Cis-NP in vivo using a SKOV3-luc cells’ xenograft model in mice.MethodsCell cytotoxicity was assessed after the exposure of the cell culture to cisplatin or Cis-NP. The effect of treatments on EMT and CSC-like phenotype was studied by analyzing a panel of markers by flow cytometry. Intracellular platinum concentration was determined by inductively coupled plasma mass spectrometry (ICS-MS), and gene expression was evaluated by RNAseq analysis. The efficacy of intraperitoneal chemotherapy was evaluated in a SKOV3-luc cells’ xenograft model in mice, through a combination of bioluminescence imaging, histological, and immunohistochemical analyses.ResultsWe observed in vitro that short-term treatment of cisplatin has a critical role in determining the potential induction of chemoresistance, and a nanotechnology-based drug delivery system can modulate it. The RNAseq analysis underlines a protective effect of nanoparticle system according to their ability to down-regulate several genes involved in chemoresistance, cell proliferation, and apoptosis. The highest intracellular platinum concentration obtained with Cis-NP treatment significantly improved the efficacy. Consistent with in vitro results, we found that Cis-NP treatment in vivo can significantly reduce tumor burden and aggressiveness compared to the free drug.ConclusionNanoparticle-mediated cisplatin delivery may serve as an intracellular depot impacting the cisplatin pharmacodynamic performance at cellular levels. These features may contribute to improving the drawbacks of conventional intraperitoneal therapy, and therefore will require further investigations in vivo.

The expression of copper transporters associated with the ototoxicity induced by platinum-based chemotherapeutic agents

BackgroundAntitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients’ quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. MethodsWe studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2′,7’ –dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. ResultsCTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79 μM, compared to 611.7 μM for L-OHP and 882.9 μM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 μM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. ConclusionsThe results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.

Immobilisation of Platinum by Cupriavidus metallidurans

The metal resistant bacterium Cupriavidus metallidurans CH34, challenged with aqueous platinous and platinic chloride, rapidly immobilized platinum. XANES/EXAFS analysis of these reaction systems demonstrated that platinum binding shifted from chloride to carboxyl functional groups within the bacteria. Pt(IV) was more toxic than Pt(II), presumably due to the oxidative stress imparted by the platinic form. Platinum immobilisation increased with time and with increasing concentrations of platinum. From a bacterial perspective, intracellular platinum concentrations were two to three orders of magnitude greater than the fluid phase, and became saturated at almost molar concentrations in both reaction systems. TEM revealed that C. metallidurans was also able to precipitate nm-scale colloidal platinum, primarily along the cell envelope where energy generation/electron transport occurs. Cells enriched in platinum shed outer membrane vesicles that were enriched in metallic, colloidal platinum, likely representing an important detoxification strategy. The formation of organo-platinum compounds and membrane encapsulated nanophase platinum, supports a role for bacteria in the formation and transport of platinum in natural systems, forming dispersion halos important to metal exploration.

Spatial and temporal profile of cisplatin delivery by ultrasound-assisted intravesical chemotherapy in a bladder cancer model.

Non-muscle invasive bladder cancer is one of the most common tumors of the urinary tract. Despite the current multimodal therapy, recurrence and progression of disease have been challenging problems. We hereby introduced a new approach, ultrasound-assisted intravesical chemotherapy, intravesical instillation of chemotherapeutic agents and microbubbles followed by ultrasound exposure. We investigated the feasibility of the treatment for non-muscle invasive bladder cancer. In order to evaluate intracellular delivery and cytotoxic effect as a function to the thickness, we performed all experiments using a bladder cancer mimicking 3D culture model. Ultrasound-triggered microbubble cavitation increased both the intracellular platinum concentration and the cytotoxic effect of cisplatin at the thickness of 70 and 122 μm of the culture model. The duration of enhanced cytotoxic effect of cisplatin by ultrasound-triggered microbubble cavitation was approximately 1 hr. Based on the distance and duration of delivery, we further tested the feasibility of repetition of the treatment. Triple treatment increased the effective distance by 1.6-fold. Our results clearly showed spatial and temporal profile of delivery by ultrasound-triggered microbubble cavitation in a tumor-mimicking structure. Furthermore, we demonstrated that the increase in intracellular concentration results in the enhancement of the cytotoxic effect in a structure with the certain thickness. Repetition of ultrasound exposure would be treatment of choice in future clinical application. Our results suggest ultrasound-triggered microbubble cavitation can be repeatable and is promising for the local control of non-muscle invasive bladder cancer.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Association between polymorphisms in CTR1, CTR2, ATP7A, and ATP7B and platinum resistance in epithelial ovarian cancer.

The copper transporters <italic>CTR1</italic>, <italic>CTR2</italic>, <italic>ATP7A</italic>, and <italic>ATP7B</italic> regulate intracellular concentration of platinum by mediating its uptake and efflux in cells. We sought to explore the effect of genetic polymorphisms in <italic>CTR1</italic>, <italic>CTR2</italic>, <italic>ATP7A</italic>, and <italic>ATP7B</italic> on platinum resistance in patients suffering from epithelial ovarian cancer (EOC). A total of 152 Chinese EOC patients were enrolled in this study, all of whom underwent adjuvant chemotherapy using platinum and taxane after maximal debulking surgery. In total, 11 single-nucleotide polymorphisms (SNPs) in <italic>CTR1, CTR2, ATP7A</italic>, and <italic>ATP7B</italic> were genotyped in these patients. The <italic>CTR1</italic> rs10981694 polymorphism was observed to be associated with carboplatin resistance, while patients with the rs10981694 G allele showed a significantly higher rate of carboplatin resistance (OR=4.00, 95% CI 1.309-12.23, p<0.01). In addition, we found that <italic>ATP7A</italic> rs2227291 was associated with cisplatin resistance and that carriers of the C allele were more sensitive to cisplatin (OR=0.40, 95% CI: 0.17-0.94, p=0.03). Our findings suggest that the <italic>CTR1</italic> and <italic>ATP7A</italic> genetic polymorphisms could affect platinum resistance. The <italic>CTR1</italic> and <italic>ATP7A</italic> genes might be considered a predictive marker for carboplatin and cisplatin resistance, respectively. .

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Organic cation transporter OCT6 mediates cisplatin uptake and resistance to cisplatin in lung cancer.

The purposes of this study were to determine whether organic cation transporters (OCTs) can mediate platinum uptake, and whether OCT down-regulation confers resistance against cisplatin (CDDP) in cancer cells. Two lung cancer cell lines, PC-6 and PC-14, and their CDDP-resistant derivatives, PC-6/CDDP and PC-14/CDDP, were analyzed. OCT expression levels were assayed using quantitative RT-PCR and Western blotting. Additionally, the effect of OCT6 overexpression, induced by transfection of the OCT6 gene SLC22A16 using a forced expression vector, on cellular sensitivity to CDDP and on intracellular platinum accumulation was measured using PC-14/CDDP cells. Both gene and protein expression of OCT6 were decreased in both CDDP-resistant cell lines compared with their expression in their respective parental cells. Intracellular accumulation of platinum was decreased in PC-14/CDDP cells compared with the parental cells after CDDP treatment. Furthermore, OCT6 overexpression induced by transfection of the OCT6 gene (SLC22A16) forced expression vector-sensitized PC-14/CDDP cells to CDDP and oxaliplatin (L-OHP) concomitant with increased intracellular concentration of platinum. OCT6 is a mediator of platinum uptake in cancer cells, and down-regulation of OCT6 is possibly one of the mechanisms of resistance against cisplatin in lung cancer.

Assessing the intracellular concentration of platinum in medulloblastoma cell lines after Cisplatin incubation

Two different analytical approaches, external calibration and isotope dilution analysis both using flow-injection inductively coupled plasma mass spectrometry, have been developed and applied to determine the intracellular platinum concentration after Cisplatin incubation of two different medulloblastoma cell lines (UW228 and DAOY). As the internal or isotopically enriched standard was already used for cell lysis, maximum accuracy of the results was obtained, whereas a new home-built and inert injection system dramatically lowered carry-over effects and analyte loss. With limits of the detection well below 0.4μgL−1 and typical relative standard deviations of 2%, a strong correlation between the cell viability in MTT assays and the incorporated amount of Pt could be shown, which was subsequently normalized to the protein content of the samples. DAOY cells did significantly ingest more Pt and showed a higher mortality, which supports the fact that transporter expression needs to be taken into account in order to obtain meaningful results.

Nanoscale Drug Delivery Platforms Overcome Platinum-Based Resistance in Cancer Cells Due to Abnormal Membrane Protein Trafficking

The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogues as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells and the development and employment of nDDPs to improve cell uptake of cisplatin.

Upregulated expression of FGF13/FHF2 mediates resistance to platinum drugs in cervical cancer cells

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

Tissue Platinum Concentration and Tumor Response in Non–Small-Cell Lung Cancer

Platinum resistance is a major limitation in the treatment of advanced non-small-cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed the effects of tissue platinum concentrations on response and survival in NSCLC. We measured total platinum concentrations by flameless atomic absorption spectrophotometry in 44 archived fresh-frozen NSCLC specimens from patients who underwent surgical resection after neoadjuvant platinum-based chemotherapy. Tissue platinum concentration was correlated with percent reduction in tumor size on post- versus prechemotherapy computed tomography scans. The relationship between tissue platinum concentration and survival was assessed by univariate and multicovariate Cox proportional hazards regression model analysis and Kaplan-Meier analysis. Tissue platinum concentration correlated significantly with percent reduction in tumor size (P < .001). The same correlations were seen with cisplatin, carboplatin, and all histology subgroups. Furthermore, there was no significant impact of potential variables such as number of cycles and time lapse from last chemotherapy on platinum concentration. Patients with higher platinum concentration had longer time to recurrence (P = .034), progression-free survival (P = .018), and overall survival (P = .005) in the multicovariate Cox model analysis after adjusting for number of cycles. This clinical study established a relationship between tissue platinum concentration and response in NSCLC. It suggests that reduced platinum accumulation might be an important mechanism of platinum resistance in the clinical setting. Further studies investigating factors that modulate intracellular platinum concentration are warranted.

Mechanism of cisplatin resistance in human urothelial carcinoma cells

An isogenic pair of cisplatin-susceptible (NTUB1) and -resistant (NTUB1/P) human urothelial carcinoma cell lines was used to elucidate the mechanism of cisplatin resistance. The significantly lower intracellular platinum (IP) concentration, which resulted from the decreased cisplatin uptake, was found in NTUB1/P cells. The enhancement of IP concentration did not increase the susceptibility of NTUB1/P cells to cisplatin treatment. The reduction of IP concentration as well was unable to enhance the cisplatin-resistance in susceptible NTUB1 cells. This indicated that reduction of IP concentration was not the account for the development of cisplatin resistance here. Instead, the over expression of anti-apoptotic Bcl-2, anti-oxidative heme oxygenase-1 (HO-1) and cell cycle regulator p16INK4 seemed to be more important for the gaining of cisplatin in these human urothelial carcinoma cell.

Abstract A21: Tissue platinum concentration and tumor response in non-small cell lung cancer

Abstract Background: Platinum resistance is a major limitation in the treatment of advanced non-small cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed effects of tissue platinum concentrations on tumor response, time to recurrence, progression-free survival (PFS) and overall survival (OS) in patients with early-stage NSCLC following neoadjuvant platinum-based chemotherapy. Methods: We measured total platinum concentrations in 44 archived fresh frozen NSCLC specimens from patients who received neoadjuvant platinum-based chemotherapy. Samples were analyzed by flameless atomic absorption spectrophotometry to assess absorbance reading associated with platinum concentration. Four specimens from patients who underwent surgery only and one patient who received pemetrexed as neoadjuvant chemotherapy were analyzed as negative controls. Absorbance value per mg of tissue was correlated with percent reduction in tumor size on post- vs prechemotherapy CT scans. Kaplan-Meier curves and log-rank tests were used to evaluate differences in time to recurrence and survival between two groups divided by median platinum concentration. Results: Platinum absorbance values in 44 neoadjuvant specimens were easily detectable and ranged from 0.6 to 6.7 × 10−3 per mg of tissue while five negative controls demonstrated absorbance readings similar to 0.1N HCL. Platinum absorbance correlated significantly with percent reduction in tumor size (R2 = 0.48, P&amp;lt;0.00001). The same correlations were seen in cisplatin (P = 0.0003), carboplatin (P = 0.0013), adenocarcinoma (P = 0.0003) and squamous cell carcinoma (P = 0.019) groups. Furthermore, there was no significant impact of potential variables such as the type of platinum compound, number of cycles, pre-treatment tumor diameter and time lapse from last chemotherapy on platinum concentration. Patients with lower Pt concentration also had shorter time to recurrence (P = 0.0494), progression free survival (P = 0.0366) and overall survival (P = 0.035). Conclusion: This is the first tissue-based study to establish a relationship between tissue platinum concentrations and response in NSCLC. Reduced intratumoral platinum accumulation may constitute a significant mechanism of platinum resistance even in clinical specimens. Further studies investigating factors that modulate intracellular platinum concentration are warranted (Supported by National Foundation of Cancer Research 90088436, Department of Defense W81XWH-07-1-0306, National Institute of Health CA127263, CA160687 and CA16672).

CBP501-Calmodulin Binding Contributes to Sensitizing Tumor Cells to Cisplatin and Bleomycin

CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G(2) checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G(2) checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G(2)-M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G(2)-M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G(2)-M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca(2+). Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K(d) = 4.62 × 10(-8) mol/L) that is reversed by Ca(2+), whereas the K(d) for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca(2+) independent. We conclude that CaM inhibition contributes to CBP501's activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.

Abstract 2635: CBP501-calmodulin binding contributes to sensitizing tumor cells to CDDP and BLM

Abstract CBP501 is an anti-cancer drug candidate currently under the Phase II clinical trials on non-small cell lung carcinoma (NSCLC) and malignant pleural mesotherioma (MPM). CBP501 was originally described as a unique G2 checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, MAPKAP-K2, or C-Tak1. However, unlike a G2 checkpoint inhibitor, CBP501 clearly enhances the G2/M accumulation induced by cisplatin (CDDP) or bleomycin (BLM) at low dose and short exposure to tumor cells. It does not affect the accumulation at G2/M by radiation, doxorubicin or 5-FU treatment in the same condition. Our recent findings point to an additional mechanis of action (MOA) for CBP501. The enhanced G2/M accumulation by CDDP results from increased intracellular platinum concentration, which leads to increased binding of platinum to DNA. The observed CBP501-induced platinum accumulation is negated in the presence of excess Ca2+. Some calmodulin (CaM) inhibitors behave similarly to CBP501. But the therapeutic window of CBP501 was broader than CaM inhibitors. Furthermore, analysis by surface plasmon resonance (SPR) reveals a direct, high-affinity molecular interaction between CBP501 and CaM (Kd=4.62×10−8M) that is reversed by Ca2+, whereas the Kd for the complex between CBP501 and 14-3-3 is some 10-fold weaker and is Ca2+-independent. We conclude that calmodulin inhibition plays important role in the activity of CBP501 in sensitizing cancer cells to CDDP or BLM. This report presents the prospects for the development of a new class of anti-cancer drugs through the addition of unique calmodulin inhibition. 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 2635. doi:10.1158/1538-7445.AM2011-2635

Establishment of three cisplatin-resistant endometrial cancer cell lines using two methods of cisplatin exposure

Using the endometrial cancer cell line EI established in our department, we attempted to establish cisplatin (CDDP)-resistant cell lines by incremental exposure and high concentration exposure methods. Three CDDP-resistant cell lines were isolated, which could be distinguished by morphological differences. 1. Upon acquiring CDDP resistance, the cells tended to become small and grow in a floating state. This tendency was especially marked when using incremental exposure method. Using the incremental exposure method, a cell line obtained by isolating and culturing only adherent cells was designated EICR-Ia, and a cell line established by culturing only floating cells was designated EICR-If. A cell line obtained by the high concentration exposure method was designated EICR-II. 2. Upon acquiring CDDP resistance, tumor markers such as TPA and LDH increased, while proliferative capability of the cells was lowered. 3. The invasion capability was diminished in EICR-If cells, but was increased in EICR-Ia and EICR-II cells. 4. Following exposure to CDDP, the intracellular platinum concentrations were markedly elevated in EI and EICR-If cells, whereas the increase was mild in EICR-Ia and EICR-II cells and the concentration was lower than that in parent EI cells. 5. Studies of drug resistance gene expression revealed increased expression of MDR1, GSTπ, and Topo-II in EICR-If cells; increased expression of GSTπ in EICR-II cells; but no expression of any of the genes in EICR-Ia cells. 6. Analyses of cancer- and apoptosis-related genes showed increased expressions of Bcl-2, c-Myc, p53, and ICE in EICR-If cells. 7. Upon acquiring CDDP resistance, sensitivity to mitomycin and adriamycin decreased, but sensitivity to etoposide and 5-fluorouracil increased. The findings indicate that the mechanisms of CDDP resistance are different in the three cell lines.

Effect of Extracellular ATP on Cisplatin-Induced Cytotoxicity in Human Ovarian Carcinoma Cells

Background: Cisplatin resistance has been mainly associated with decreased cellular accumulation and increased intracellular glutathione (GSH) levels. ATP is known to increase the membrane permeability of cells and to decrease intracellular GSH levels. Our study aimed at using extracellular ATP to sensitize ovarian carcinoma cells towards cisplatin. Methods: The MTT assay was used to determine the EC₅₀ of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant variant A2780cis. Intracellular platinum concentrations were determined using flameless atomic absorption spectrometry. Results: Preincubation and concomitant incubation with ATP did not alter the EC₅₀ of cisplatin. The presence of 100 µM ATP along with cisplatin decreased cell survival in A2780 but not in A2780cis cells. Extracellular ATP did not increase the cellular accumulation of cisplatin in both A2780 and A2780cis cells. Conclusion: The presence of extracellular ATP during treatment with cisplatin leads to additive cytotoxicity in the sensitive A2780 but not in cisplatin-resistant A2780cis cells.