Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer

BackgroundCisplatin (DDP) is the first-in-class drug for advanced and non-targetable non-small-cell lung cancer (NSCLC). A recent study indicated that DDP could slightly induce non-apoptotic cell death ferroptosis, and the cytotoxicity was promoted by ferroptosis inducer. The agents enhancing the ferroptosis may therefore increase the anticancer effect of DDP. Several lines of evidence supporting the use of phytochemicals in NSCLC therapy. Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, showed anticancer effects on NSCLC by triggering autophagy. Ferroptosis can be triggered by autophagy, which regulates redox homeostasis. Thus, we aimed to elucidate the possible role of ferroptosis involved in the synergistic effect of ginkgetin and DDP in cancer therapy.MethodsThe promotion of DDP-induced anticancer effects by ginkgetin was examined via a cytotoxicity assay and western blot. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blot, and qPCR. With ferroptosis blocking, the contribution of ferroptosis to ginkgetin + DDP-induced cytotoxicity, the Nrf2/HO-1 axis, and apoptosis were determined via a luciferase assay, immunostaining, chromatin immunoprecipitation (CHIP), and flow cytometry. The role of ferroptosis in ginkgetin + DDP-treated NSCLC cells was illustrated by the application of ferroptosis inhibitors, which was further demonstrated in a xenograft nude mouse model.ResultsGinkgetin synergized with DDP to increase cytotoxicity in NSCLC cells, which was concomitant with increased labile iron pool and lipid peroxidation. Both these processes were key characteristics of ferroptosis. The induction of ferroptosis mediated by ginkgetin was further confirmed by the decreased expression of SLC7A11 and GPX4, and a decreased GSH/GSSG ratio. Simultaneously, ginkgetin disrupted redox hemostasis in DDP-treated cells, as demonstrated by the enhanced ROS formation and inactivation of the Nrf2/HO-1 axis. Ginkgetin also enhanced DDP-induced mitochondrial membrane potential (MMP) loss and apoptosis in cultured NSCLC cells. Furthermore, blocking ferroptosis reversed the ginkgetin-induced inactivation of Nrf2/HO-1 as well as the elevation of ROS formation, MMP loss, and apoptosis in DDP-treated NSCLC cells.ConclusionThis study is the first to report that ginkgetin derived from Ginkgo biloba leaves promotes DDP-induced anticancer effects, which can be due to the induction of ferroptosis.     

How to teach an old dog new tricks: Autophagy-independent action of chloroquine on the tumor vasculature

Chloroquine (CQ) is exploited in clinical trials as an autophagy blocker to potentiate anticancer therapy, but it is unknown if it solely acts by inhibiting cancer cell-autonomous autophagy. Our recent study shows that besides blocking cancer cell growth, CQ also affects endothelial cells (ECs) and promotes tumor vessel normalization. This vessel normalizing effect of CQ reduces tumor hypoxia, cancer cell intravasation, and metastasis, while improving the delivery and response to chemotherapy. By compromising autophagy in melanoma cells or using mice with a conditional knockout of ATG5 in ECs, we found that the favorable effects of CQ on the tumor vasculature do not rely on autophagy. CQ-induced vessel normalization relies mainly on altered endolysosomal trafficking and sustained NOTCH1 signaling in ECs. Remarkably these CQ-mediated effects are abrogated when tumors are grown in mice harboring EC-specific deletion of NOTCH1. The autophagy-independent vessel normalization by CQ leading to improved delivery and tumor response to chemotherapy further advocates its clinical use in combination with anticancer treatments.     

How to teach an old dog new tricks: Autophagy-independent action of chloroquine on the tumor vasculature

Chloroquine (CQ) is exploited in clinical trials as an autophagy blocker to potentiate anticancer therapy, but it is unknown if it solely acts by inhibiting cancer cell-autonomous autophagy. Our recent study shows that besides blocking cancer cell growth, CQ also affects endothelial cells (ECs) and promotes tumor vessel normalization. This vessel normalizing effect of CQ reduces tumor hypoxia, cancer cell intravasation, and metastasis, while improving the delivery and response to chemotherapy. By compromising autophagy in melanoma cells or using mice with a conditional knockout of ATG5 in ECs, we found that the favorable effects of CQ on the tumor vasculature do not rely on autophagy. CQ-induced vessel normalization relies mainly on altered endolysosomal trafficking and sustained NOTCH1 signaling in ECs. Remarkably these CQ-mediated effects are abrogated when tumors are grown in mice harboring EC-specific deletion of NOTCH1. The autophagy-independent vessel normalization by CQ leading to improved delivery and tumor response to chemotherapy further advocates its clinical use in combination with anticancer treatments.     

Biodegradable thermosensitive hydrogel for SAHA and DDP delivery: therapeutic effects on oral squamous cell carcinoma xenografts

Background

OSCC is one of the most common malignancies and numerous clinical agents currently applied in combinative chemotherapy. Here we reported a novel therapeutic strategy, SAHA and DDP-loaded PECE (SAHA-DDP/PECE), can improve the therapeutic effects of intratumorally chemotherapy on OSCC cell xenografts.

Objective/Purpose

The objective of this study was to evaluate the therapeutic efficacy of the SAHA-DDP/PECE in situ controlled drug delivery system on OSCC cell xenografts.

Methods

A biodegradable and thermosensitive hydrogel was successfully developed to load SAHA and DDP. Tumor-beared mice were intratumorally administered with SAHA-DDP/PECE at 50 mg/kg (SAHA) +2 mg/kg (DDP) in 100 ul PECE hydrogel every two weeks, SAHA-DDP at 50 mg/kg(SAHA) +2 mg/kg(DDP) in NS, 2 mg/kg DDP solution, 50 mg/kg SAHA solution, equal volume of PECE hydrogel, or equal volume of NS on the same schedule, respectively. The antineoplastic actions of SAHA and DDP alone and in combination were evaluated using the determination of tumor volume, immunohistochemistry, western blot, and TUNEL analysis.

Results

The hydrogel system was a free-flowing sol at 10°C, become gel at body temperature, and could sustain more than 14 days in situ. SAHA-DDP/PECE was subsequently injected into tumor OSCC tumor-beared mice. The results demonstrated that such a strategy as this allows the carrier system to show a sustained release of SAHA and DDP in vivo, and could improved therapeutic effects compared with a simple additive therapeutic effect of SAHA and DDP on mouse model.

Conclusions

Our research indicated that the novel SAHA-DDP/PECE system based on biodegradable PECE copolymer enhanced the therapeutic effects and could diminished the side effects of SAHA/DDP. The present work might be of great importance to the further exploration of the potential application of SAHA/DDP-hydrogel controlled drug release system in the treatment of OSCC.     

Chloroquine Enhances Gefitinib Cytotoxicity in Gefitinib-Resistant Nonsmall Cell Lung Cancer Cells

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, these patients eventually develop resistance to EGFR-TKI. The goal of the present study was to investigate the involvement of autophagy in gefitinib resistance. We developed gefitinib-resistant cells (PC-9/gef) from PC-9 cells (containing exon 19 deletion EGFR) after long-term exposure in gefitinib. PC-9/gef cells (B4 and E3) were 200-fold more resistant to gefitinib than PC-9/wt cells. Compared with PC-9/wt cells, both PC-9/gefB4 and PC-9/gefE3 cells demonstrated higher basal LC3-II levels which were inhibited by 3-methyladenine (3-MA, an autophagy inhibitor) and potentiated by chloroquine (CQ, an inhibitor of autophagolysosomes formation), indicating elevated autophagy in PC-9/gef cells. 3-MA and CQ concentration-dependently inhibited cell survival of both PC-9wt and PC-9/gef cells, suggesting that autophagy may be pro-survival. Furthermore, gefitinib increased LC3-II levels and autolysosome formation in both PC-9/wt cells and PC-9/gef cells. In PC-9/wt cells, CQ potentiated the cytotoxicity by low gefitinib (3nM). Moreover, CQ overcame the acquired gefitinib resistance in PC-9/gef cells by enhancing gefitinib-induced cytotoxicity, activation of caspase 3 and poly (ADP-ribose) polymerase cleavage. Using an in vivo model xenografting with PC-9/wt and PC-9/gefB4 cells, oral administration of gefitinib (50 mg/kg) completely inhibited the tumor growth of PC-9/wt but not PC-9/gefB4cells. Combination of CQ (75 mg/kg, i.p.) and gefitinib was more effective than gefitinib alone in reducing the tumor growth of PC-9/gefB4. Our data suggest that inhibition of autophagy may be a therapeutic strategy to overcome acquired resistance of gefitinib in EGFR mutation NSCLC patients.     

Inhibition of Autophagy Potentiated the Antitumor Effect of Nedaplatin in Cisplatin-Resistant Nasopharyngeal Carcinoma Cells

Nedaplatin, a cisplatin analog, was developed to reduce the toxicity of cisplatin, whereas it can be cross-resistant with cisplatin in some circumstances. This study aimed to investigate the role of autophagy in nedaplatin induced cell death in cisplatin-resistant nasopharyngeal carcinoma cells. Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity. Nedaplatin treatment resulted in autophagosome accumulation and increased expression of LC3-II, indicating the induction of autophagy by nedaplatin in HNE1/DDP and CNE2/DDP cells. Inhibition of autophagy by Bafilomycin A1 (Baf A1) and 3-Methyladenine (3-MA) remarkably enhanced the antitumor efficacy of nedaplatin in HNE1/DDP and CNE2/DDP cells, suggesting that the resistance to nedaplatin-induced cell death was caused by enhanced autophagy in nedaplatin-resistant NPC cells. Additionally, Baf A1 enhanced reactive oxygen species (ROS) generation and apoptosis induced by nedaplatin in HNE1/DDP cells. Mechanistically, nedaplatin treatment caused activation of ERK1/2 and suppression of Akt/mTOR signaling pathways. While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells. Furthermore, suppression of ROS could inhibit nedaplatin-induced ERK activation in HNE1/DDP cells, indicating that ROS and ERK were involved in nedaplatin-induced autophagy. Together, these findings suggested that autophagy played a cytoprotective role in nedaplatin-induced cytotoxicity of HNE1/DDP and CNE2/DDP cells. Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.     

Lobetyolin suppressed lung cancer in a mouse model by inhibiting epithelial-mesenchymal transition

Traditional Chinese medicines are gaining more attention as promising adjuvant agents for conventional chemotherapy. Recent studies have shown that lobetyolin (LBT) is one of the main bioactive compounds of traditional Chinese medicines and it exhibits anticancer activity in several types of cancer. Therefore, this study aimed to investigate the mechanism by which LBT inhibits lung cancer. A549 human lung cancer cells were treated with LBT. In addition, A549 cells were injected into Balc/b nude mice to establish model of lung cancer. The mice were treated with cisplatin (DDP) or LBT alone or in combination, and tumor growth was monitored. Protein levels of E-cadherin, vimentin and matrix metalloproteinase 9 (MMP9) were detected. We found that the combination of LBT and DDP showed stronger effect to inhibit the proliferation of A549 cells compared to LBT or DDP treatment alone. Wound healing assay showed that the ratio of wound healing was significantly lower in LBT group and DDP group and was the lowest in LBT+DDP group. Transwell invasion assay showed that the invasion ability of A549 cells was the weakest in LBT+DDP group. Protein levels of E-cadherin were the highest while those of vimentin and MMP9 were the lowest in A549 cells treated with LBT+DDP. Nude mouse xenograft tumor model showed that the combination of LBT with DDP had the highest efficacy to inhibit the growth of lung cancer, and tumor tissues of mice treated with LBT+DDP had the lowest expression of vimentin and MMP9 and the highest expression of E-cadherin. In conclusion, LBT significantly enhances the efficacy of chemotherapy on lung cancer, and the mechanism may be related to the inhibition of epithelial-mesenchymal transition.Key words: Lobetyolin, lung cancer, cisplatin, epithelial-mesenchymal transition, Chinese traditional medicine     

富含miR-30a的牛初乳组分通过抑制癌细胞自噬促进顺铂化疗的肿瘤消亡

本实验首次发现牛初乳中富含miR-30a.取牛初乳中富含miR-30a的组分处理用顺铂化疗的癌细胞,能显著地抑制癌细胞自噬,促进癌细胞凋亡:单独使用顺铂化疗18 h的癌细胞凋亡率为12.5%,而化疗的同时施加富含miR-30a的牛初乳组分18 h后癌细胞凋亡率为56.5%.用这种富含miR-30a的牛初乳组分饲喂顺铂化疗的荷瘤小鼠,能更有效地抑制肿瘤生长:化疗小鼠饲喂牛初乳组分后,肿瘤直径自6 mm缩小为4.5 mm,而单独用顺铂化疗的肿瘤直径约8 mm.因此富含miR-30a的牛初乳组分能增进化疗药物的疗效.施用牛初乳组分,还可减少化疗药物的用量:单独用顺铂处理细胞18 h需20 mg/L剂量,加牛初乳组分后10 mg/L即可取得明显凋亡效果;单独用顺铂化疗荷瘤小鼠10天每次需500μg,而添加富含miR-30a的牛初乳组分后,顺铂剂量减半即可明显缩小肿瘤.     

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.     

精脒与疟原虫抗氯喹关系的研究

抑制伯氏疟原虫感染RBC中50％精脒量,抗氯喹株需要喂服400mg/kg氯喹,而敏感的为20mg/kg,相差至少20倍。两株感染鼠同样喂服氟喹20mg/kg和400mg/kg一次,3小时后,在全血和感染RBC中的氯喹量,低剂量组接近,400mg/kg组抗氯喹的更高,而积聚在疟原虫中的氯喹量则抗氯喹的低于敏感。抗氯喹感染RBC中的精脒,78‰集中在疟原虫,故认为精脒量的提高,可能与氯喹进入疟原虫产生竞争性抑制。MGBG抑制抗氯喹鼠中的疟原虫生长,精脒能逆转其作用。     

细胞自噬在吉非替尼治疗非小细胞肺癌中的作用及其机制研究

目的:观察吉非替尼对非小细胞肺癌细胞自噬的影响,并探讨其机制。方法:将BalB/CA-nu品系裸小鼠分成两组,各组20只,均造肺癌模型,其中吉非替尼治疗组小鼠在肺癌组模型的基础上给予吉非替尼25mg/kg 14d,停药后剖杀。取组织切片,通过免疫荧光、RT-PCR以及Western blot的试验方法检测自噬相关基因Beclin1和MAPLC3的表达。结果:免疫荧光、RT-PCR以及Western blot的试验方法检测均发现肺癌组小鼠组织中Beclin1和MAPLC3表达较低,而在吉非替尼处理组中,Beclin1和MAPLC3的表达明显升高,差异有统计学意义。结论:吉非替尼可以通过增强细胞自噬从而发挥对非小细胞肺癌的抑制作用。     

Nutritional and supranutritional levels of selenate differentially suppress prostate tumor growth in adult but not young nude mice

The inhibitory effect of oral methylseleninic acid or methylselenocysteine administration on cancer cell xenograft development in nude mice is well characterized; however, less is known about the efficacy of selenate and age on selenium chemoprevention. In this study, we tested whether selenate and duration on diets would regulate prostate cancer xenograft in nude mice. Thirty-nine homozygous NU/J nude mice were fed a selenium-deficient, Torula yeast basal diet alone (Se-) or supplemented with 0.15 (Se) or 1.0 (Se+) mg selenium/kg (as Na(2)SeO(4)) for 6 months in Experiment 1 and for 4 weeks in Experiment 2, followed by a 47-day PC-3 prostate cancer cell xenograft on the designated diet. In Experiment 1, the Se- diet enhanced the initial tumor development on days 11-17, whereas the Se+ diet suppressed tumor growth on days 35-47 in adult nude mice. Tumors grown in Se- mice were loosely packed and showed increased necrosis and inflammation as compared to those in Se and Se+ mice. In Experiment 2, dietary selenium did not affect tumor development or histopathology throughout the time course. In both experiments, postmortem plasma selenium concentrations in Se and Se+ mice were comparable and were twofold greater than those in Se- mice. Taken together, dietary selenate at nutritional and supranutritional levels differentially inhibit tumor development in adult, but not young, nude mice engrafted with PC-3 prostate cancer cells.     

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rg^null mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.     

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100–120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1–15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5×10⁴ Cetus units twice daily for 4–5 days), 5-FU augmented (up to 37-fold, days 1–9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3–4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3–9), as determined by limiting-dilution cultures with IL-2 (for 7–8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6–9 days after treatment with 5-FU, Adriamycin or dacarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6–12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1-LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.     

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.     

Chloroquine Is Grossly Under Dosed in Young Children with Malaria: Implications for Drug Resistance

Background

Plasmodium falciparum malaria is treated with 25 mg/kg of chloroquine (CQ) irrespective of age. Theoretically, CQ should be dosed according to body surface area (BSA). The effect of dosing CQ according to BSA has not been determined but doubling the dose per kg doubled the efficacy of CQ in children aged <15 years infected with P. falciparum carrying CQ resistance causing genes typical for Africa. The study aim was to determine the effect of age on CQ concentrations.

Methods and Findings

Day 7 whole blood CQ concentrations were determined in 150 and 302 children treated with 25 and 50 mg/kg, respectively, in previously conducted clinical trials. CQ concentrations normalised for the dose taken in mg/kg of CQ decreased with decreasing age (p<0.001). CQ concentrations normalised for dose taken in mg/m² were unaffected by age. The median CQ concentration in children aged <2 years taking 50 mg/kg and in children aged 10–14 years taking 25 mg/kg were 825 (95% confidence interval [CI] 662–988) and 758 (95% CI 640–876) nmol/l, respectively (p = 0.67). The median CQ concentration in children aged 10–14 taking 50 mg/kg and children aged 0–2 taking 25 mg/kg were 1521 and 549 nmol/l. Adverse events were not age/concentration dependent.

Conclusions

CQ is under-dosed in children and should ideally be dosed according to BSA. Children aged <2 years need approximately double the dose per kg to attain CQ concentrations found in children aged 10–14 years. Clinical trials assessing the efficacy of CQ in Africa are typically performed in children aged <5 years. Thus the efficacy of CQ is typically assessed in children in whom CQ is under dosed. Approximately 3 fold higher drug concentrations can probably be safely given to the youngest children. As CQ resistance is concentration dependent an alternative dosing of CQ may overcome resistance in Africa.     

红景天苷调节AMPK/mTOR/ULK1信号通路对结肠癌SW480细胞裸鼠的肝脏损伤的影响

摘要 目的：探讨红景天苷（Sal）调节单磷酸腺苷活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）/Unc51样激酶1（ULK1）信号通路对结肠癌SW480细胞裸鼠肝脏损伤的影响。方法：通过皮下注射SW480细胞悬浮液建立肝转移裸鼠模型,将造模后的裸鼠随机分为模型组、Sal低剂量（Sal-L,50 mg/kg Sal）组、Sal中剂量（Sal-M,100 mg/kg Sal）组、Sal高剂量（Sal-H,200 mg/kg Sal）组,Sal-H+AMPK抑制剂（Compound C,200 mg/kg Sal+10 mg/kg Compound C）组,以未接种SW480细胞悬液的裸鼠作为对照组。腹部主动脉取血,检测裸鼠血清中丙氨酸氨基转移酶（AST）、天冬氨酸氨基转移酶（ALT）水平;处死裸鼠,检测肝转移瘤数目及肝脏重量;HE染色观察肝脏组织病理变化;qRT-PCR检测肝脏组织中AMPK、mTOR、ULK1 mRNA表达水平;Western blot检测肝脏组织中自噬（Beclin1、p62）蛋白及通路相关蛋白表达。结果：与对照组相比,模型组裸鼠组织中出现肝转移瘤,肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）;与模型组相比,Sal-L、Sal-M、Sal-H组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著降低（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著增加（P<0.05）;与Sal-H组相比,Sal-H+Compound C组肝转移瘤数目、肝脏重量、AST、ALT水平、mTORmRNA、ULK1 mRNA、p62表达显著增加（P<0.05）;Beclin1、AMPK mRNA及蛋白表达显著降低（P<0.05）。结论：Sal可通过减少裸鼠肝转移瘤形成,保护裸鼠肝脏,其机制可能与激活AMPK/mTOR/ULK1信号通路,促进肝脏自噬有关。     

Polyphyllin I induced-apoptosis is enhanced by inhibition of autophagy in human hepatocellular carcinoma cells

BackgroundPolyphyllin I (PPI), a bioactive phytochemical isolated from the rhizoma of Paris polyphyllin, exerts preclinical anticancer efficacy in various cancer models. However, the effects of PPI on regulatory human hepatocellular carcinoma (HCC) cell proliferation and its underlying mechanisms remain unknown.PurposeThis study investigated the antiproliferation effect of PPI on HCC cells and its underlying mechanisms.MethodsCell viability was measured by MTT assay. Cell death, apoptosis and acidic vesicular organelles (AVOs) formation were determined by flow cytometry. Protein levels were analyzed by Western blot analysis.ResultsPPI induced apoptosis through the caspase-dependent pathway and activated autophagy through the PI3K/AKT/mTOR pathway. Blockade of autophagy by pharmacological inhibitors or RNA interference enhanced the cytotoxicity and antiproliferation effects of PPI. Moreover, chloroquine (CQ) enhanced the antiproliferation effect of PPI on HCC cells via the caspase-dependent apoptosis pathway by inhibiting protective autophagy. Therefore, the combination therapy of CQ and PPI exhibited synergistic effects on HCC cells compared with CQ or PPI alone.ConclusionThe current findings strongly indicate that PPI can induce protective autophagy in HCC cells, thereby providing a novel target in potentiating the anticancer effects of PPI and other chemotherapeutic drugs in liver cancer treatment. Moreover, the combination therapy of CQ and PPI is an effective and promising candidate to be further developed as therapeutic agents in the treatment of liver cancer.