Intracellular Pharmacokinetics of Activated Drugs in a Prodrug-Enzyme-Ultrasound System: Evaluations on ZD2767P+CPG2+US.

Intracellular pharmacokinetics (PK) of activated drugs is a window to understanding the pharmacodynamics of prodrug-enzyme-ultrasound therapy. Herein PK of ZD2767D (i.e., activated drug) in the ZD2767P+CPG2+US system on A549, A549/DDP, SKOV3, and SKOV3/DDP cells were evaluated (A549/DDP and SKOV3/DDP were cisplatin-resistant sublines). The noncompartment model under extravascular input mode was deemed appropriate for evaluating drug level vs time curves; Cmax, AUClast, MRTlast, Vz, and Cl can reflect the PK feature, but t1/2, AUCinf, and MRTinf were irrational; higher accumulation and slower elimination characterized the PK mechanism of ZD2767P+CPG2+US; enhanced permeability and retention effect can be assessed with Cmax, AUClast, MRTlast, and tlast; ultrasound equivalently modulated Cmax and AUClast in sensitive and resistant cells. The experimental design and dose proportionality were discussed.

Clinical Trials of High-Intensity Focused Ultrasound for Cancer: Concerns Arising from Low Post-Hoc Power.

Prospective controlled trials of high-intensity focused ultrasound for cancers were evaluated. Post-hoc power was <0.80 in 30/47 trials and in 22/39 trials with positive results, indicating low quality in most trials. Unscientific endpoints, small sample sizes, and high dropout rates led to low post-hoc power that caused inter-trial heterogeneity and overestimated the therapeutic effect. The objective response rate was not a substitute for survival time for estimating the sample size and assessing the efficacy. The present data can interpret a paradox: HIFU is considered to have slighter cytotoxicity to non-cancer tissues and no radiation but is frequently combined with chemotherapy and/or radiotherapy in practice.

Metabolomic profiling identifies hair as a robust biological sample for identifying women with cervical cancer.

Metabolomics serves as a useful tool for identifying biomarkers of disease and uncovering pathogenic mechanisms. However, most metabolomic studies use biological fluids such as blood and urine as biospecimens, which could be dramatically influenced by daily activities and dietary variation, resulting in measurement fluctuations. In contrast, hair may serve as a robust source of stable longitudinal metabolite information. Here, we conducted a pilot study to investigate the possibility of using hair as a biospecimen for the metabolomic analysis of cervical cancer. Hair, plasma, urine, and cervical tissue samples from cervical cancer and benign tumor patients were collected. Biospecimens were then tested using a gas chromatography-mass spectrometry-based metabolomic platform. The expressions of enzymatic genes related to metabolic changes were validated using qPCR. Statistical analyses were calculated via the R-console platform. Metabolite profiles in both hair and cervical tissue samples were significantly different between cancer and control groups, while no difference was observed in plasma and urine samples. Further analysis showed that most of the altered metabolites in hair were upregulated, and they had a negative correlation with those in the cervical tissue. Eight common metabolites showed an area under the Receiver Operating Characteristic curve greater than 0.95. These metabolites primarily participated in amino acid metabolism, cofactor synthesis, ferroptosis, and glycolysis. The gene expressions (IDH1, OGDH, GLUD1, ENO1, GSS, and GPX4) associated with the shortlisted metabolic pathways were also upregulated. Our study is the first to reveal metabolomic changes of hair in cervical cancer patients and demonstrates the potential for the hair metabolome to be used for biomarker identification in cervical cancer.

Ultrasonically Enhanced ZD2767P-Carboxypeptidase G2 Deactivates Cisplatin-Resistant Human Lung Cancer Cells.

The prodrug-enzyme regimen ZD2767P+CPG2 is limited by low efficacy. Here, ultrasound was used to modulate ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against cisplatin-resistant human lung cancer cells. A549 and A549/DDP (resistant subline) cells received ZD2767P+CPG2 or ZD2767P+CPG2+US. Either ZD2767P+CPG2 or ZD2767P+CPG2+US led to cell death and apoptosis, and ZD2767P+CPG2+US produced stronger effects; comet assays revealed that these two means directly caused DNA double-strand break. Z-VAD-fmk and/or ferrostatin-1 increased the cell survival percentage, and Z-VAD-fmk decreased the apoptosis percentage. The level of transferrin was increased in treated cells, but those of ferroportin and glutathione peroxidase 4 (GPX4) were reduced, with higher intracellular levels of reactive oxygen species and of iron. Intracellular pharmacokinetics of ZD2767D (activated drug) indicated that the peak level, area under the drug level vs. time curve, and mean residence time in ZD2767P+CPG2+US were higher than those in ZD2767P+CPG2. Both ZD2767P+CPG2 and ZD2767P+CPG2+US were effective on xenograft tumors in nude mice; inhibitory rates were 39.7% and 63.5% in A549 tumors and 50.0% and 70.1% in A549/DDP tumors, respectively. A higher apoptosis level and a lower GPX4 level were noted in tumors receiving treatments. No severe adverse events were observed. These data demonstrated that ZD2767P+CPG2+US deactivated cancer cells via apoptosis and ferroptosis pathways, being a candidate therapy for cisplatin-resistant lung cancer.

IDO1 Modulates the Sensitivity of Epithelial Ovarian Cancer Cells to Cisplatin through ROS/p53-Dependent Apoptosis.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing dioxygenase that may play a part in chemoresistance in ovarian cancer. However, its role in cisplatin (DDP) resistance is unclear. Here, the expression level of IDO1 in tumors in platinum-resistant (n = 22) and -sensitive (n = 46) ovarian cancer patients was determined, and then how IDO1 modulated DDP resistance was explored in vitro and in vivo. The IDO1 expression level in platinum-resistant patients was higher than that in -sensitive patients, and a higher IDO1 level was correlated with poor prognosis in type II cancer patients. Up-regulating IDO1 decreased DDP-induced apoptosis in SKOV3 cells via inhibiting the ROS/p53 cell-death pathway, thereby attenuating cytotoxicity of DDP. Silencing IDO1 enhanced p53-dependent apoptosis by increasing ROS accumulation, thereby enhancing DDP against SKOV3 cells. Down-knocking IDO1 augmented the action of DDP in vivo. These data demonstrated that silencing IDO1 enhanced the efficacy of DDP by intensifying p53-dependent apoptosis, and that targeting IDO1 can be a strategy to modulate DDP-based chemotherapy for epithelial ovarian cancer.

Complex metabolic interactions between ovary, plasma, urine, and hair in ovarian cancer.

Ovarian cancer (OC) is the third most common malignant tumor of women accompanied by alteration of systemic metabolism, yet the underlying interactions between the local OC tissue and other system biofluids remain unclear. In this study, we recruited 17 OC patients, 16 benign ovarian tumor (BOT) patients, and 14 control patients to collect biological samples including ovary plasma, urine, and hair from the same patient. The metabolic features of samples were characterized using a global and targeted metabolic profiling strategy based on Gas chromatography-mass spectrometry (GC-MS). Principal component analysis (PCA) revealed that the metabolites display obvious differences in ovary tissue, plasma, and urine between OC and non-malignant groups but not in hair samples. The metabolic alterations in OC tissue included elevated glycolysis (lactic acid) and TCA cycle intermediates (malic acid, fumaric acid) were related to energy metabolism. Furthermore, the increased levels of glutathione and polyunsaturated fatty acids (linoleic acid) together with decreased levels of saturated fatty acid (palmitic acid) were observed, which might be associated with the anti-oxidative stress capability of cancer. Furthermore, how metabolite profile changes across differential biospecimens were compared in OC patients. Plasma and urine showed a lower concentration of amino acids (alanine, aspartic acid, glutamic acid, proline, leucine, and cysteine) than the malignant ovary. Plasma exhibited the highest concentrations of fatty acids (stearic acid, EPA, and arachidonic acid), while TCA cycle intermediates (succinic acid, citric acid, and malic acid) were most concentrated in the urine. In addition, five plasma metabolites and three urine metabolites showed the best specificity and sensitivity in differentiating the OC group from the control or BOT groups (AUC > 0.90) using machine learning modeling. Overall, this study provided further insight into different specimen metabolic characteristics between OC and non-malignant disease and identified the metabolic fluctuation across ovary and biofluids.

Thioridazine Enhances Cisplatin-Induced DNA Damage in Cisplatin-Resistant Human Lung Cancer Cells.

Thioridazine was used to sensitize cisplatin against cisplatin-resistant human lung cancer cells. Cells received thioridazine, cisplatin, or both drugs (the combination). Thioridazine synergized cisplatin to increase percentages of dead and apoptotic cells. DNA damage was detected using the comet assays; the combination led to the highest alkaline- and neutral-comet percentages, demonstrating exacerbation of both single- and double-strand breaks. After thioridazine treatment, levels of glutathione, and BRCA2, RAD51, and ERCC1 proteins were decreased. These data manifested that thioridazine decreased the capacities of detoxification and DNA repair, thereby enhancing cisplatin-induced DNA damage in resistant cells.

NTNG1 Modulates Cisplatin Resistance in Epithelial Ovarian Cancer Cells via the GAS6/AXL/Akt Pathway.

Cisplatin resistance is a challenge in the treatment of epithelial ovarian cancer. Here, clinical data showed that the level of netrin-G1 (NTNG1) in cisplatin-resistant cancer was higher than that in cisplatin-sensitive cancer (2.2-fold, p = 0.005); patients with a high NTNG1 level in cancer tissues had shorter progression-free survival (11.0 vs. 25.0 months, p = 0.010) and platinum-free interval (5.0 vs. 20.0 months, p = 0.021) compared with patients with a low level. Category- or stage-adjusted analyses demonstrated that the association between the NTNG1 level and prognosis occurred in type II or FIGO III/IV cancer. The basal level of NTNG1 in SKOV3/DDP cells (a cisplatin-resistant subline) was higher than that in SKOV3 cells; therefore, NTNG1 was overexpressed in SKOV3 cells, or silenced in SKOV3/DDP cells. Knocking in NTNG1 reduced the action of cisplatin to decrease cell death and apoptosis of SKOV3 cells, accompanied by upregulation of p-AXL, p-Akt and RAD51; however, opposite effects were observed in SKOV3/DDP cells after knocking down NTNG1. Co-immunoprecipitation demonstrated that NTNG1 bound GAS6/AXL. Silencing NTNG1 enhanced cisplatin effects in vivo, decreasing tumor volume/mass. These data suggested that a high NTNG1 level can result in cisplatin resistance in ovarian cancer cells via the GAS6/AXL/Akt pathway and that NTNG1 may be a useful target to overcome resistance.

Erratum: DR2 blocker thioridazine: A promising drug for ovarian cancer therapy.

[This corrects the article DOI: 10.3892/ol.2017.7184.].

Ultrasound Enhances ZD2767P-Carboxypeptidase G2 against Chemoresistant Ovarian Cancer Cells by Altering the Intracellular Pharmacokinetics of ZD2767D.

Prodrug-carboxypeptidase G2 (e.g., ZD2767P+CPG2) can realize a targeted treatment where the specific advantage is a lack of CPG2 analogues in humans, but it is limited by low efficacy. Here ultrasound was employed to enhance ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against chemoresistant human ovarian cancer cells. The release dynamics of ZD2767D (activated drug) by CPG2 were investigated. The in vitro efficacy was explored in SKOV3 and SKOV3/DDP (cisplatin-resistant subline) cells; spectrophotometry was established to quantify ZD2767P and ZD2767D, and then intracellular pharmacokinetics were evaluated. The in vivo efficacy was validated in both subcutaneous and orthotopic tumors. With insonation, the ZD2767D concentration was increased during an early period. Insonation synergized ZD2767P+CPG2 to enhance cell death and apoptosis, and efficacies in SKOV3 and SKOV3/DDP cells were similar. Intracellular pharmacokinetics of ZD2767D were nonproportional, and insonation increased the peak level, area under the level vs time curve, and mean residence time. In subcutaneous xenografts, ZD2767P+CPG2 and ZD2767P+CPG2+US resulted in volume-inhibitory rates of 20.4% and 26.5% in SKOV3 tumors and 36.8% and 81.6% in SKOV3/DDP tumors, respectively. In the orthotopic tumor model, the survival time in group ZD2767P+CPG2 or ZD2767P+CPG2+US was prolonged compared with group control, in SKOV3 (33.0 ± 3.5 or 39.2 ± 1.8 vs 25.0 ± 1.6 days, p < 0.0001) and SKOV3/DDP (16.2 ± 4.8 or 22.3 ± 7.3 vs 8.7 ± 3.9 days, p = 0.0015) tumors. These data indicated that ZD2767P+CPG2+US was effective against resistant ovarian cancer cells.

Erratum: DR2 blocker thioridazine: A promising drug for ovarian cancer therapy.

[This corrects the article DOI: 10.3892/ol.2017.7184.].

Thioridazine Sensitizes Cisplatin Against Chemoresistant Human Lung and Ovary Cancer Cells.

Human lung cancer cell lines A549 and A549/DDP, and ovarian cancer cell lines SKOV3 and SKOV3/DDP were subjected to thioridazine (Thio), cisplatin, or the combination; A549/DDP and SKOV3/DDP were the cisplatin-resistant sublines. Cell viability, apoptosis, and cell cycle were detected; the mitochondrial membrane potential and proteins related to mitochondrial apoptosis were determined. Thio induced cell death, and the combination of Thio and cisplatin led to the highest percentage of dead cells in four cells lines. Thio and the combined modality led to cell apoptosis by inducing G0/G1 arrest. The collapse of mitochondrial membrane potential, activation of caspase 9, upregulation of Bax protein, and downregulation of Bcl-2 protein demonstrated that apoptosis was mitochondria dependent. These data indicated that Thio could be used to modulate cisplatin-based chemotherapeutic regimen in lung and ovary cancers.

miR-27b-3p/MARCH7 regulates invasion and metastasis of endometrial cancer cells through Snail-mediated pathway.

Ubiquitin E3 ligase membrane-associated RING-CH-type finger 7 (MARCH7), also known as axotrophin, was originally identified in mouse embryonic stem cells. MARCH7 is involved in T-cell proliferation, neuronal development, and the immune system. However, its role in endometrial cancer (EC) remains unclear. This study aimed to investigate the role of MARCH7 in EC. Quantitative polymerase chain reaction, immunohistochemistry, and western blot analysis were used to examine the expression of MARCH7, E-cadherin, Snail, and Vimentin in EC cell lines or clinical specimens. The role of MARCH7 in maintaining EC cell malignant phenotype was determined by transwell assay and using xenograft tumor model. Dual-luciferase reporter gene assay was performed to determine whether MARCH7 is an authentic target of miR-27b-3p. Our data showed that the expression level of MARCH7 in EC tissues was higher than that in normal endometrium tissues. The level of MARCH7 was positively associated with that of Snail and Vimentin, clinical stage, and histological grade, while negatively associated with that of E-cadherin. Knockdown of MARCH7 inhibited the invasion and metastasis of EC cells in vitro and in vivo. The opposite effect was observed after overexpressing MARCH7. MARCH7 promoted invasion and metastasis of EC cells via the Snail-mediated pathway. Furthermore, MARCH7 was demonstrated to be an authentic target of miR-27b-3p, and miR-27b-3p decreased the stimulus effect induced by MARCH7. These data indicate that MARCH7 may be an oncogenic factor and a therapeutic target for EC. miR-27b-3p/MARCH7 may also regulate EC cell invasion and metastasis via the Snail-mediated pathway.

Linc02527 promoted autophagy in Intrahepatic cholestasis of pregnancy.

LncRNA plays a crucial role in human disease. However, the expression and function of LncRNA in ICP(Intrahepatic cholestasis of pregnancy) is still not fully elucidated. In this study, we found Linc02527 was increased expression in placenta and serum of ICP patients. Ectopically expression of Linc02527 promoted autophagy and proliferate in HTR8 cells. Silencing Linc02527 suppressed the autophagy and proliferate in HTR8 cells. Mechanically study revealed that Linc02527 regulated the expression of ATG5 and ATG7 by sponging miR-3185. Linc02527 directly binding to YBX1 and activated P21. The growth of C57 mouse was retarded when autophagy was activated. In normal condition, inhibited autophagy using chloroquine did not affect the growth of C57 mouse. However, in the condition of autophagy was activated, inhibited autophagy using chloroquine can improve the growth of C57 mouse. Overall, the results of this study identified Linc02527 as a candidate biomarker in ICP and a potential target for ICP therapy. Chloroquine was a potential drug for ICP therapy.

Nanosecond Electric Pulses Induce Early and Late Phases of DNA Damage and Cell Death in Cisplatin-Resistant Human Ovarian Cancer Cells.

Chemoresistance is a challenge for management of ovarian cancer, and therefore the response of resistant cells to nanosecond electric pulses (nsEP) was explored. Human ovarian cancer cell line COC1 and the cisplatin-resistant subline COC1/DDP were subjected to nsEP (32 ns, 10 kV/cm, 10 Hz pulse repletion frequency, and 10 min exposure duration), and then the cellular responses were followed. The percentages of dead cells and of comet-formed cells in the alkaline assay displayed two peak levels (i.e., 2 and 8 h after nsEP exposure), with the highest value noted at 8 h; the percentage of comet-formed cells in the neutral assay was increased at 8 h; the apoptotic percentage was increased at 8 h, with collapse of the mitochondrial membrane potential and the activation of caspase-3 and caspase-9. The comet assay demonstrated DNA single-strand break at 2 h and double-strand break at 8 h. nsEP resulted in lower cytotoxicity in COC1/DDP cells compared with COC1 cells. These findings indicated that nsEP induced early and late phases of DNA damage and cell death, and these two types of cell death may have distinct applications to treatments of chemoresistant ovarian cancers.

Photodynamic Therapy Mediated by Aloe-Emodin Inhibited Angiogenesis and Cell Metastasis Through Activating MAPK Signaling Pathway on HUVECs.

Photodynamic therapy is a clinically used, minimally invasive therapeutic procedure that involves the application of photosensitizers which can locate in target cells and so be irradiated at a corresponding wavelength. Laser light irradiation activation of photosensitizers generates free reactive oxygen species, which induces selective cytotoxic activity in target cells. Within recent years, aloe-emodin as a photosensitizer has been successfully applied in photodynamic therapy applications. Angiogenesis plays an important role in tumor growth and metastasis; thus, the development of a novel target treatment for angiogenesis is essential in order to improve treatment therapeutics for cancer treatment. An essential step in angiogenesis involves the formation of tube-like structures during matrix degradation, rearrangement, and apoptosis of endothelial cells. In the present study, we investigated the mechanisms of photocytotoxicity induced by aloe-emodin in human umbilical vein endothelial cells. Analysis of cell proliferation results noted a significant decrease in cultured cells which received various concentrations of aloe-emodin and photodynamic therapy-induced light doses. Additionally, mitochondrial mechanisms of apoptotic cell death were observed in aloe-emodin photodynamic therapy-treated cells, as tube formation assays noted angiogenesis suppression after treatment. The capacity of migration and invasion of human umbilical vein endothelial cells was measured using the transwell assay and demonstrated that aloe-emodin photodynamic therapy significantly inhibited the migration and invasion of human umbilical vein endothelial cells. The expression of p38, extracellular signal-regulated kinase, the c-Jun N-terminal kinases, and vascular endothelial growth factor suggested that the cellular metastasis was related to mitogen-activated protein kinase signal pathway. Furthermore, disorganization of F action cytoskeleton components was observed after aloe-emodin photodynamic therapy. Overall, the findings from this study suggest that aloe-emodin photodynamic therapy inhibited angiogenesis and cellular metastasis in human umbilical vein endothelial cells by activating the mitogen-activated protein kinase apoptotic signaling cell death pathway.

Insulin Treatment Cannot Promote Lipogenesis in Rat Fetal Lung in Gestational Diabetes Mellitus Because of Failure to Redress the Imbalance Among SREBP-1, SCAP, and INSIG-1.

Gestational diabetes mellitus (GDM) has a higher incidence of neonatal respiratory distress syndrome, and lipogenesis is required for the synthesis of pulmonary surfactants. The aim of this study was to determine the effect of insulin treatment in GDM on the production of lipids in the lungs of fetal rats. GDM was induced by streptozotocin, and insulin was used to manage diabetes. Type II alveolar epithelial cells (AEC II), bronchoalveolar lavage fluid (BALF), and lung tissues of the neonatal rats were sampled for analyses. Insulin treatment could not decrease plasma glucose to normal level at a later gestational stage. Lipids/phospholipids in AEC II, BALF, and lung tissues decreased in GDM, and insulin treatment could not increase the levels; quantitative PCR and western blotting demonstrated a lower level of sterol regulator element-binding protein 1 (SREBP-1), SREBP cleavage-activating protein (SCAP), and insulin-induced gene 1 (INSIG-1) in GDM, but insulin treatment upregulated only SREBP-1. Nuclear translocation of the SREBP-1 protein in AEC II was impaired in GDM, which could not be ameliorated by insulin treatment. These findings indicated that insulin treatment in GDM cannot promote lipogenesis in the fetal lung because of failure to redress the imbalance among SREBP-1, SCAP, and INSIG-1.

Knockdown of BRCA2 enhances cisplatin and cisplatin-induced autophagy in ovarian cancer cells.

Clinical implications of the BRCA2 expression level on treatments of ovarian cancer are controversial. Here, we demonstrated that platinum-resistant cancer had a higher percentage of high BRCA2 level (87.5% vs 43.6%, P = 0.001), and that patients with a low BRCA2 level in cancer tissues had longer progression-free survival (with a median time of 28.0 vs 12.0 months, P < 0.001) and platinum-free duration (with a median time of 19.0 vs 5.0 months, P < 0.001) compared with those with a high BRCA2 level. In human ovarian cancer cell lines CAOV-3 and ES-2, cisplatin induced an upregulation of the RAD51 protein, which was inhibited after silencing BRCA2; silencing BRCA2 enhanced the action of cisplatin in vitro and in vivo Knockdown of BRCA2 promoted cisplatin-induced autophagy. Interestingly, the autophagy blocker chloroquine enhanced cisplatin in BRCA2-silenced cells accompanied by an increase in apoptotic cells, which did not occur in BRCA2-intact cells; chloroquine enhanced the efficacy of cisplatin against BRCA2-silenced CAOV-3 tumors in vivo, with an increase in LC3-II level in tumor tissues. Sensitization of cisplatin was also observed in BRCA2-silenced CAOV-3 cells after inhibiting ATG7, confirming that chloroquine modulated the sensitivity via the autophagy pathway. These data suggest that a low BRCA2 level can predict better platinum sensitivity and prognosis, and that the modulation of autophagy can be a chemosensitizer for certain cancers.

Deactivation of cisplatin-resistant human lung/ovary cancer cells with pyropheophorbide-α methyl ester-photodynamic therapy.

The aim of this study was to determine whether photodynamic therapy (PDT) alone or combined with cisplatin (DDP), can deactivate cisplatin-resistant cancer cells. Human cancer cell lines A549 and SKOV3, and chemoresistant sublines A549/DDP and SKOV3/DDP, were subjected to PDT, DDP, or PDT combined with DDP. Cell viability and apoptosis were analyzed, and then intracellular reactive oxygen species (ROS) and proteins related to apoptosis were determined. PDT caused cell death, and PDT combined with DDP led to the highest percentage of dead cells in 4 cell lines; similar results were detected in ROS; a quantification evaluation manifested that the combined effect was addition. DDP increased the percentage of apoptotic cells, and the ROS level in A549 and SKOV3 cells, which was not observed in A549/DDP and SKOV3/DDP cells. Western blot revealed an increase of caspase 3 and Bax, and a decrease of Bcl-2, demonstrating the occurrence of apoptosis. The data suggest that PDT can efficiently deactivate resistant cells and enhance the action of DDP against resistant cancer cells.