KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model.
ABSTRACT: Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.
Project description:The large-conductance Ca<sup>2+</sup> -activated K<sup>+</sup> channel K<sub>Ca</sub> 1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three-dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). K<sub>Ca</sub> 1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. K<sub>Ca</sub> 1.1 activator-induced hyperpolarizing responses were significantly larger in human osteosarcoma MG-63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of K<sub>Ca</sub> 1.1 and its role in chemoresistance using a 3D spheroid model. K<sub>Ca</sub> 1.1 protein expression levels were significantly elevated in the lipid-raft-enriched compartments of MG-63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to K<sub>Ca</sub> 1.1 protein degradation in breast cancer. The siRNA-mediated inhibition of FBXW7 in MG-63 cells from 2D monolayers upregulated K<sub>Ca</sub> 1.1 protein expression. Furthermore, a treatment with a potent and selective K<sub>Ca</sub> 1.1 inhibitor overcame the chemoresistance of the MG-63 and human chondrosarcoma SW-1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP-binding cassette transporters, the expression of the multidrug resistance-associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of K<sub>Ca</sub> 1.1. Therefore, the pharmacological inhibition of K<sub>Ca</sub> 1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of K<sub>Ca</sub> 1.1-positive sarcomas.
Project description:Modulation of endothelial calcium-activated K<sup>+</sup> channels has been proposed as an approach to restore arterial endothelial cell function in disease. We hypothesized that small-conductance calcium-activated K<sup>+</sup> channels (K<sub>Ca</sub>2.3 or SK3) contributes to erectile function. The research was performed in transgenic mice with overexpression (K<sub>Ca</sub>2.3 <sup>T/T(-Dox)</sup>) or down-regulation (K<sub>Ca</sub>2.3 <sup>T/T(+Dox)</sup>) of the K<sub>Ca</sub>2.3 channels and wild-type C57BL/6-mice (WT). QPCR revealed that K<sub>Ca</sub>2.3 and K<sub>Ca</sub>1.1 channels were the most abundant in mouse corpus cavernosum. K<sub>Ca</sub>2.3 channels were found by immunoreactivity and electron microscopy in the apical-lateral membrane of endothelial cells in the corpus cavernosum. Norepinephrine contraction was enhanced in the corpus cavernosum of K<sub>Ca</sub>2.3 <sup>T/T(+Dox)</sup> versus K<sub>Ca</sub>2.3 <sup>T/T(-Dox)</sup> mice, while acetylcholine relaxation was only reduced at 0.3?µM and relaxations in response to the nitric oxide donor sodium nitroprusside were unaltered. An opener of K<sub>Ca</sub>2 channels, NS309 induced concentration-dependent relaxations of corpus cavernosum. Mean arterial pressure was lower in K<sub>Ca</sub>2.3 <sup>T/T(-Dox)</sup> mice compared with WT and K<sub>Ca</sub>2.3 <sup>T/T(+Dox)</sup> mice. In anesthetized mice, cavernous nerve stimulation augmented in frequency/voltage dependent manner erectile function being lower in K<sub>Ca</sub>2.3 <sup>T/T(+Dox)</sup> mice at low frequencies. Our findings suggest that down-regulation of K<sub>Ca</sub>2.3 channels contributes to erectile dysfunction, and that pharmacological activation of K<sub>Ca</sub>2.3 channels may have the potential to restore erectile function.
Project description:Prostate cancer (PCa) is the second most common cancer type in men, and in advanced metastatic stages is considerable incurable. This justifies the need for efficient early diagnostic methods and novel therapies, particularly radiopharmaceuticals with the potential for simultaneous diagnosis and therapy (theranostics). We have previously demonstrated, using monolayer-cultured cells, that copper-64 chloride, a promising theranostic agent for PCa, has the potential to induce significant damage in cancer cells while having minimal side effects in healthy tissues. Here, we further explored this compound for its theranostic applications using more advanced PCa cellular models, specifically multicellular spheroids. Namely, we evaluated the cellular uptake of <sup>64</sup>CuCl<sub>2</sub> in three human PCa spheroids (derived from 22RV1, DU145, and LNCaP cells), and characterized the growth profile and viability of those spheroids as well as the clonogenic capacity of spheroid-derived cells after exposure to <sup>64</sup>CuCl<sub>2</sub>. Furthermore, the populations of cancer stem cells (CSCs), known to be important for cancer resistance and recurrence, present in the spheroid models were also evaluated using two different markers (CD44 and CD117). <sup>64</sup>CuCl<sub>2</sub> was found to have significant detrimental effects in spheroids and spheroid-derived cells, being able to reduce their growth and impair the viability and reproductive ability of spheroids from both castration-resistant (22RV1 and DU145) and hormone-naïve PCa (LNCaP). Interestingly, resistance to <sup>64</sup>CuCl<sub>2</sub> treatment seemed to be related with the presence of a CSC population, since the most resistant spheroids, derived from the DU145 cell line, had the highest initial percentage of CSCs among the three cell lines under study. Altogether, these results clearly highlight the theranostic potential of <sup>64</sup>CuCl<sub>2</sub>.
Project description:Cholic acid-conjugated methylcellulose-polyethylenimines (MCPEI-CAs) were synthesized and characterized for drug delivery systems. Their synthesis was confirmed by ¹H NMR and FT-IR analysis. Induced circular dichroism result with Congo red showed that methylcellulose (MC) and polyethylenimine-grafted cationic derivative (MC-PEI) would have helical conformation and random coil structure, respectively. It was found that MCPEI-CAs could form positively charged (>30 mV Zeta-potential) and spherical nano-aggregates (~250 nm Z-average size) by hydrophobic interaction of CA moieties. Critical aggregation concentration of MCPEI-CA<sub>10</sub> was measured as 7.2 × 10<sup>-3</sup> mg/mL. MCPEI-CA<sub>10</sub> could encapsulate the anticancer drug doxorubicin (Dox) with 58.0% of drug loading content and 23.2% of drug loading efficiency and its release was facilitated in acidic condition. Cytotoxicity of MCPEI-CAs was increased with the increase of cholic acid (CA) graft degrees, probably due to the cellular membrane disruption by interaction with specific molecular structure of amphiphilic MCPEI-CA nano-aggregates. MCPEI-CA<sub>10</sub>/Dox nano-aggregates showed concentration-dependent anticancer activity, which could overcome the multidrug resistance of cancer cells. In this work, molecular conformation change of MC derivatives by chemical modification and a potential of MCPEI-CA<sub>10</sub>/Dox nano-aggregates for drug delivery systems were revealed.
Project description:<h4>Background and purpose</h4>Doxorubicin (DOX) is an effective cancer therapeutic agent but causes therapy-limiting cardiotoxicity. The effects of DOX and its metabolite doxorubicinol (DOXL) on individual channels have been well characterized in isolation. However, it is unknown how the action and interaction of affected channels combine to generate the phenotypic cardiotoxic outcome. We sought to develop an in silico model that links drug effects on channels to action potential duration (APD) and intracellular Ca<sup>2+</sup> concentration in order to address this gap in knowledge.<h4>Experimental approach</h4>We first propose two methods to obtain, from published values, consensus drug effects on the currents of individual channels, transporters and pumps. Separately, we obtained equivalent values for APD and Ca<sup>2+</sup> concentration (the readouts used as surrogates for cardiotoxicity). Once derived, the consensus effects on the currents were incorporated into established biophysical models of the cardiac myocyte and were refined adjusting the sarcoplasmic reticulum Ca<sup>2+</sup> leak current (I<sub>Leak</sub> ) until the consensus effects on APD and Ca<sup>2+</sup> dynamics were replicated. Using factorial analysis, we then quantified the relative contribution of each channel to DOX and DOXL cardiotoxicity.<h4>Key results</h4>The factorial analysis identified the rapid delayed rectifying K<sup>+</sup> current, the L-type Ca<sup>2+</sup> current and the sarcoplasmic reticulum I<sub>Leak</sub> as the targets primarily responsible for the cardiotoxic effects on APD and Ca<sup>2+</sup> dynamics.<h4>Conclusions and implications</h4>This study provides insight into the mechanisms of DOX-induced cardiotoxicity and a framework for the development of future diagnostic and therapeutic strategies.
Project description:<h4>Background</h4>Combined therapy has demonstrated to be an effective strategy for cancer therapy. Herein, an injectable hydrogel based on the genetically engineered polypeptide and hollow gold nanoshells (HAuNS) has been developed for chemo-photothermal therapy of HepG2 tumor.<h4>Methods</h4>PC<sub>10</sub>A/DOX/HAuNS nanogel was prepared with layer-by-layer through the adsorption of DOX and PC<sub>10</sub>A successively. DOX with positive charge and PC<sub>10</sub>A with negative charge were coated step by step onto the surface of negatively charged HAuNS. The multifunctional hydrogel PC<sub>10</sub>A/DOX/HAuNS were prepared via dissolving hybrid PC<sub>10</sub>A/DOX/HAuNS nanogel in polypeptide PC<sub>10</sub>A. Chemotherapy drug DOX in the PC<sub>10</sub>A/DOX/HAuNS hydrogel was absorbed on the HAuNS and directly embedded in the PC<sub>10</sub>A hydrogel, which contributes to sequentially release of the drug. Specifically, DOX adsorbed on the HAuNS could be released slowly for sustainable chemotherapy.<h4>Results</h4>The PC<sub>10</sub>A/DOX/HAuNS hydrogel could pass 26-gauge needle without clogging, indicating that it is injectable. In addition, the PC<sub>10</sub>A/DOX/HAuNS hydrogel possessed outstanding photothermal effect and photothermal stability. In both in vitro cell and in vivo tumor-bearing mice experiments, a remarkably enhance tumor inhibition was observed by the combined therapy of chemo-photothermal therapy compared with photothermal therapy or chemotherapy alone.<h4>Conclusions</h4>The combined chemotherapy and photothermal therapy of PC<sub>10</sub>A/DOX/HAuNS hydrogels could significantly improve the therapeutic effect. Therefore, the multifunctional hydrogel PC<sub>10</sub>A/DOX/HAuNS is promising to provide a new strategy for sustained chemo-photothermal therapy.
Project description:Contactin 1 (CNTN1) is a new oncogenic protein of prostate cancer (PC); its impact on PC remains incompletely understood. We observed CNTN1 upregulation in LNCaP cell-derived castration-resistant PCs (CRPC) and CNTN1-mediated enhancement of LNCaP cell proliferation. CNTN1 overexpression in LNCaP cells resulted in enrichment of the CREIGHTON_ENDOCRINE_THERAPY_RESISTANCE_3 gene set that facilitates endocrine resistance in breast cancer. The leading-edge (LE) genes (<i>n</i> = 10) of this enrichment consist of four genes with limited knowledge on PC and six genes novel to PC. These LE genes display differential expression during PC initiation, metastatic progression, and CRPC development, and they predict PC relapse following curative therapies at hazard ratio (HR) 2.72, 95% confidence interval (CI) 1.96-3.77, and <i>p</i> = 1.77 × 10<sup>-9</sup> in The Cancer Genome Atlas (TCGA) PanCancer cohort (<i>n</i> = 492) and HR 2.72, 95% CI 1.84-4.01, and <i>p</i> = 4.99 × 10<sup>-7</sup> in Memorial Sloan Kettering Cancer Center (MSKCC) cohort (<i>n</i> = 140). The LE gene panel classifies high-, moderate-, and low-risk of PC relapse in both cohorts. Additionally, the gene panel robustly predicts poor overall survival in clear cell renal cell carcinoma (ccRCC, <i>p</i> = 1.13 × 10<sup>-11</sup>), consistent with ccRCC and PC both being urogenital cancers. Collectively, we report multiple CNTN1-related genes relevant to PC and their biomarker values in predicting PC relapse.
Project description:<b>Background and Purpose:</b> Doxorubicin (DOX) is a risk factor for arm lymphedema in breast cancer patients. We reported that DOX opens ryanodine receptors (RYRs) to enact "calcium leak," which disrupts the rhythmic contractions of lymph vessels (LVs) to attenuate lymph flow. Here, we evaluated whether dantrolene, a clinically available RYR1 subtype antagonist, prevents the detrimental effects of DOX on lymphatic function. <b>Experimental Approach:</b> Isolated rat mesenteric LVs were cannulated, pressurized (4-5 mm Hg) and equilibrated in physiological salt solution and Fura-2AM. Video microscopy recorded changes in diameter and Fura-2AM fluorescence tracked cytosolic free calcium ([Ca<sup>2+</sup> <sub>i</sub>]). High-speed <i>in vivo</i> microscopy assessed mesenteric lymph flow in anesthetized rats. Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs). <b>Key Results:</b> DOX (10 μmol/L) increased resting [Ca<sup>2+</sup> <sub>i</sub>] by 17.5 ± 3.7% in isolated LVs (<i>n</i> = 11). The rise in [Ca<sup>2+</sup> <sub>i</sub>] was prevented by dantrolene (3 μmol/L; n = 10). A single rapid infusion of DOX (10 mg/kg i.v.) reduced positive volumetric lymph flow to 29.7 ± 10.8% (<i>n</i> = 7) of baseline in mesenteric LVs <i>in vivo</i>. In contrast, flow in LVs superfused with dantrolene (10 μmol/L) only decreased to 76.3 ± 14.0% (<i>n</i> = 7) of baseline in response to DOX infusion. Subsequently, expression of the RYR1 subtype protein as the presumed dantrolene binding site was confirm in isolated mesenteric LMCs by flow cytometry. <b>Conclusion and Implications:</b> We conclude that dantrolene attenuates the acute impairment of lymph flow by DOX and suggest that its prophylactic use in patients subjected to DOX chemotherapy may lower lymphedema risk.
Project description:Prostate-specific membrane antigen (PSMA) is a prospect biomarker for the treatment of prostate cancer. Meanwhile, positron emission tomography (PET) is being developed as a significant imaging modality in cancer diagnosis. A new PET probe Glu-ureido-Lys-naphthylalanine-tranexamic acid-Gly(AMBF<sub>3</sub>)-triiodobenzoic acid (<sup>18</sup>F-GLNTGT) was radiosynthesized by a one-step <sup>18</sup>F-labeled method. <sup>18</sup>F-GLNTGT was obtained with a radioactivity yield (RCY) of 12.16 ± 6.4% and a good radiochemical purity (RCP > 96%). The cell uptakes of <sup>18</sup>F-GLNTGT were determined to be 15.9 ± 0.43% ID and 9.47 ± 1.26% ID at 15 min in LNCaP cells and PC-3 cells, respectively. The cell internalization of <sup>18</sup>F-GLNTGT was determined to be 12.89 ± 0.94% ID and 5.34 ± 0.15% ID at 15 min in LNCaP cells and PC-3 cells, respectively. It is suggested that the probe has good specificity targeting PSMA. From the results of <sup>18</sup>F-GLNTGT binding affinity with PSMA, it has a higher affinity and a <i>K</i> <sub>i</sub> value of 0.49 nM (95% confidence interval (CI): 0.35-0.67 nM). In PET imaging, <sup>18</sup>F-GLNTGT showed the highest tumor uptake of 3.51 ± 0.15% ID/g at 45 min and the maximum tumor/muscle (T/M<sub>max</sub>) ratio of 3.68 ± 0.29 at 60 min post-injection (p.i.) in LNCaP tumors. The control probe <sup>18</sup>F-AlF-NOTA-RGD<sub>2</sub> presented the highest tumor uptake of 4.2 ± 0.54% ID/g at 7.5 min and the T/M<sub>max</sub> ratio of 2.72 ± 0.63 at 45 min p.i. in LNCaP tumors. The results showed that the probe has a higher tumor/muscle ratio compared with the control probe <sup>18</sup>F-AlF-NOTA-RGD<sub>2</sub>. Although the probe <sup>18</sup>F-GLNTGT has some limitations for CT signal detection both in cells and <i>in vivo</i>, it is still a promising PET probe for targeting PSMA membrane protein.
Project description:Current cancer therapies are frequently ineffective and associated with severe side effects and with acquired cancer drug resistance. The development of effective therapies has been hampered by poor correlations between pre-clinical and clinical outcomes. Cancer cell-derived spheroids are three-dimensional (3D) structures that mimic layers of tumors in terms of oxygen and nutrient and drug resistance gradients. Gold nanoparticles (AuNP) are promising therapeutic agents which permit diminishing the emergence of secondary effects and increase therapeutic efficacy. In this work, 3D spheroids of Doxorubicin (Dox)-sensitive and -resistant colorectal carcinoma cell lines (HCT116 and HCT116-DoxR, respectively) were used to infer the potential of the combination of chemotherapy and Au-nanoparticle photothermy in the visible (green laser of 532 nm) to tackle drug resistance in cancer cells. Cell viability analysis of 3D tumor spheroids suggested that AuNPs induce cell death in the deeper layers of spheroids, further potentiated by laser irradiation. The penetration of Dox and earlier spheroid disaggregation is potentiated in combinatorial therapy with Dox, AuNP functionalized with polyethylene glycol (AuNP@PEG) and irradiation. The time point of Dox administration and irradiation showed to be important for spheroids destabilization. In HCT116-sensitive spheroids, pre-irradiation induced earlier disintegration of the 3D structure, while in HCT116 Dox-resistant spheroids, the loss of spheroid stability occurred almost instantly in post-irradiated spheroids, even with lower Dox concentrations. These results point towards the application of new strategies for cancer therapeutics, reducing side effects and resistance acquisition.