Lin-Lin Chang

Baicalein Triggers Autophagy and Inhibits the Protein Kinase B/Mammalian Target of Rapamycin Pathway in Hepatocellular Carcinoma HepG2 Cells

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24‐h treatment as determined by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) assay. However, BA (50 μM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24‐h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up‐regulated the expression of microtubule‐associated protein 1A/1B‐light chain 3‐II in concentration‐dependent and time‐dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration‐dependently and time‐dependently decreased the expression of p‐AKT (S473), p‐ULK1 (S757) and p‐4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA‐triggered autophagy. Copyright

Glycyrrhetinic Acid Triggers a Protective Autophagy by Activation of Extracellular Regulated Protein Kinases in Hepatocellular Carcinoma Cells

Glycyrrhetinic acid (GA), one of the main constituents of the famous Chinese medicinal herb and food additive licorice (Glycyrrhiza uralensis Fisch), has been indicated to possess potential anticancer effects and is widely utilized in hepatocellular carcinoma (HCC) targeted drug delivery systems (TDDS) due to the highly expressed target binding sites of GA on HCC cells. This study found that GA reduced the cell viability, increased the release of lactate dehydrogenase, and enhanced the expression of Bax, cleaved caspase-3, and LC3-II in HCC cells. The GA-triggered autophagy has been further confirmed by monodansylcadaverine staining as well as transmission electron microscopy analysis. The cell viability was obviously decreased whereas the expression of cleaved caspases was significantly increased when inhibition of autophagy by choloroquine or bafilomycin A1, suggesting that GA triggered a protective autophagy. Extracellular regulated protein kinase (ERK) was activated after treatment with GA in HepG2 cells and pretreatment with U0126 or PD98059, the MEK inhibitors, reversed GA-triggered autophagy as evidenced by decreased expression of LC3-II and formation of autophagosomes, respectively. Furthermore, GA-induced cell death and apoptosis were enhanced after pretreatment with PD98059. This is the first report that GA triggers a protective autophagy in HCC cells via activation of ERK, which might attenuate the anticancer effects of GA or chemotherapeutic drugs loaded with GA-modified TDDS.

DJ-1 mediates the resistance of cancer cells to dihydroartemisinin through reactive oxygen species removal

Dihydroartemisinin (DHA), one of the main metabolites of artemisinin and its derivatives, presents anti-cancer potential in vitro and in vivo. To explore the mechanisms of resistance toward DHA, a DHA-resistant cell line, HeLa/DHA, was established with a resistance factor of 7.26 in vitro. Upon DHA treatment, apoptotic cells were significantly elicited in parental HeLa cells but minimally induced in HeLa/DHA cells. HeLa/DHA cells also displayed much less sensitivity to DHA-induced tumor suppression in cancer xenograft models than HeLa cells. Intriguingly, DHA-resistant cells did not display a multidrug-resistant phenotype. Based on a proteomic study employing LC-ESI-MS/MS together with pathway analysis, DJ-1 (PARK7) was found to be highly expressed in HeLa/DHA cells. Western blot and immunofluorescence assays confirmed the higher expression of DJ-1 in HeLa/DHA cells than in parental cells in both cell line and xenograft models. DJ-1 is translocated to the mitochondria of HeLa/DHA cells and oxidized, providing DJ-1 with stronger cytoprotection activity. Further study revealed that DJ-1 knockdown in HeLa/DHA cells abolished the observed resistance, whereas overexpression of DJ-1 endowed the parental HeLa cells with resistance toward DHA. Reactive oxygen species (ROS) were also significantly induced by either DHA or hydrogen peroxide in HeLa cells but not in resistant HeLa/DHA cells. When the cells were pretreated with N-acetyl-l-cysteine, the effect of DJ-1 knockdown on sensitizing HeLa/DHA cells to DHA was significantly attenuated. In summary, our study suggests that overexpression and mitochondrial translocation of DJ-1 provides HeLa/DHA cells with resistance to DHA-induced ROS and apoptosis.

Nuclear translocation and activation of YAP by hypoxia contributes to the chemoresistance of SN38 in hepatocellular carcinoma cells

Although hypoxia is a prominent feature contributing to the therapeutic resistance of hepatocellular carcinoma cells (HCC) against chemotherapeutic agents, including the Topoisomerase I inhibitor SN38, the underlying mechanism is not fully understood and its understanding remains a major clinical challenge. In the present study, we found that hypoxia-induced nuclear translocation and accumulation of YAP acted as a survival input to promote resistance to SN38 in HCC. The induction of YAP by hypoxia was not mediated by HIF-1α because manipulating the abundance of HIF-1α with CoCl2, exogenous expression, and RNA interference had no effect on the phosphorylation or total levels of YAP. The mevalonate-HMG-CoA reductase (HMGCR) pathway may modulate the YAP activation under hypoxia. Combined YAP inhibition using either siRNA or the HMGCR inhibitor statins together with SN38 treatment produced improved anti-cancer effects in HCC cells. The increased anti-cancer effect of the combined treatment with statins and irinotecan (the prodrug of SN-38) was further validated in a human HepG2 xenograft model of HCC in nude mice. Taken together, our findings identify YAP as a novel mediator of hypoxic-resistance to SN38. These results suggest that the administration of SN28 together with the suppression of YAP using statins is a promising strategy for enhancing the treatment response in HCC patients, particularly in advanced stage HCC cases presenting hypoxic resistance.

Platycodin D triggers autophagy through activation of extracellular signal-regulated kinase in hepatocellular carcinoma HepG2 cells

Platycodin D (PD), isolated from the Chinese medicinal herb named Platycodonis Radix, is a triterpenoid saponin with well-known anti-tumor effects. In this study, we provided reliable evidence that PD triggered autophagy in a number of cell lines in vitro. PD-triggered autophagy was identified by observation of cytoplasmic vacuole, up-regulation of microtubule-associated protein 1 light chain 3 II (LC3-II), and accumulation of autophagosomes. The Akt/mammalian target of rapamycin (mTOR) pathway may be not involved in PD-triggered autophagy, as evidenced by the increased phosphorylation of Akt (Thr308), mTOR (Ser2448), ribosomal protein S6 kinase (Ser371), and ULK1 (Ser757). However, the extracellular signal-regulated kinase (ERK) was activated after PD treatment. The decreased ERK phosphorylation caused by pretreatment with U0126, an inhibitor of MEK, suppressed the expression of LC3-II compared with PD treatment alone, suggesting that ERK pathway may have a critical function in PD-triggered autophagy. In addition, the PD-induced proliferative inhibition and apoptosis were enhanced when pretreatment with autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (BAF), indicating that PD may trigger a protective autophagy in HepG2 cells. To the best of our knowledge, this paper is the first to report that PD triggers autophagy in a series of cell lines and ERK activation is important for PD-triggered autophagy in hepatocellular carcinoma HepG2 cells. The combined treatment with PD and CQ or BAF may be a promising regimen for hepatocellular carcinoma treatment.

Inactivation of hypoxia-induced YAP by statins overcomes hypoxic resistance tosorafenib in hepatocellular carcinoma cells

Sorafenib is a multikinase inhibitor used as a first-line treatment for advanced hepatocellular carcinoma (HCC), but it has shown modest to low response rates. The characteristic tumour hypoxia of advanced HCC maybe a major factor underlying hypoxia-mediated treatment failure. Thus, it is urgent to elucidate the mechanisms of hypoxia-mediated sorafenib resistance in HCC. In this study, we found that hypoxia induced the nuclear translocation of Yes associate-Protein (YAP) and the subsequent transactivation of target genes that promote cell survival and escape apoptosis, thereby leading to sorafenib resistance. Statins, the inhibitors of hydroxymethylglutaryl-CoA reductase, could ameliorate hypoxia-induced nuclear translocation of YAP and suppress mRNA levels of YAP target genes both in vivo and in vitro. Combined treatment of statins with sorafenib greatly rescued the loss of anti-proliferative effects of sorafenib under hypoxia and improved the inhibitory effects on HepG2 xenograft tumour growth, accompanied by enhanced apoptosis as evidenced by the increased sub-G1 population and PARP cleavage. The expression levels of YAP and its target genes were highly correlated with poor prognosis and predicted a high risk of HCC patients. These findings collectively suggest that statins utilization maybe a promising new strategy to counteract hypoxia-mediated resistance to sorafenib in HCC patients.

Aldo–Keto Reductase AKR1C1–AKR1C4: Functions, Regulation, and Intervention for Anti-cancer Therapy

Aldo–keto reductases comprise of AKR1C1–AKR1C4, four enzymes that catalyze NADPH dependent reductions and have been implicated in biosynthesis, intermediary metabolism, and detoxification. Recent studies have provided evidences of strong correlation between the expression levels of these family members and the malignant transformation as well as the resistance to cancer therapy. Mechanistically, most studies focus on the catalytic-dependent function of AKR1C isoforms, like their impeccable roles in prostate cancer, breast cancer, and drug resistance due to the broad substrates specificity. However, accumulating clues showed that catalytic-independent functions also played critical roles in regulating biological events. This review summarizes the catalytic-dependent and -independent roles of AKR1Cs, as well as the small molecule inhibitors targeting these family members.

AKR1C1 Activates STAT3 to Promote the Metastasis of Non-Small Cell Lung Cancer

Metastasis is the leading cause of mortality for human non-small cell lung cancer (NSCLC). However, it is difficult to target tumor metastasis because the molecular mechanisms underlying NSCLC invasion and migration remain unclear. Methods: GEO data analyses and IHC analyses were performed to identify that the expression level of AKR1C1, a member of human aldo-keto reductase family, was highly elevated in patients with metastasis or metastatic foci of NSCLC patients. Functional analyses (in vitro and in vivo) and quantitative genomic analyses were preformed to confirm the pro-metastatic effects of AKR1C1 and the underlying mechanisms. The correlation of AKR1C1 with the prognosis of NSCLC patients was evaluated using Kaplan-Meier analyses. Results: in NSCLC patients, AKR1C1 expression was closely correlated with the metastatic potential of tumors. AKR1C1 overexpression in nonmetastatic cancer cells significantly promoted metastasis both in vitro and in vivo, whereas depletion of AKR1C1 in highly metastatic tumors potently alleviated these effects. Quantitative genomic and functional analyses revealed that AKR1C1 directly interacted with STAT3 and facilitated its phosphorylation—thus reinforcing the binding of STAT3 to the promoter regions of target genes—and then transactivated these genes, which ultimately promoted tumor metastasis. Further studies showed that AKR1C1 might facilitate the interaction of STAT3 with its upstream kinase JAK2. Intriguingly, AKR1C1 exerted these pro-metastatic effects in a catalytic-independent manner. In addition, a significant correlation between AKR1C1 and STAT3 pathway was observed in the metastatic foci of NSCLC patients, and the AKR1C1-STAT3 levels were highly correlated with a poor prognosis in NSCLC patients. Conclusions: taken together, we show that AKR1C1 is a potent inducer of NSCLC metastasis. Our study uncovers the active function of AKR1C1 as a key component of the STAT3 pathway, which promotes lung cancer metastasis, and highlights a candidate therapeutic target to potentially improve the survival of NSCLC patients with metastatic disease.

Hypoxia-Targeted Drug Q6 Induces G2-M Arrest and Apoptosis via Poisoning Topoisomerase II under Hypoxia.

In spite of the tremendous efforts dedicated to developing hypoxia-activated prodrugs, no agents yet have been approved for clinical therapy. In the present study, the hypoxic selective anti-cancer activity as well as the cellular target of a novel tirapazamine (TPZ) analogue, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (Q6) were investigated. Q6 implemented anti-cancer effects via poisoning topoisomerase II (topo II) under hypoxia. Modified trapped in agarose DNA immunostaining (TARDIS) assay showed more topo II–DNA cleavage complexes trapped by Q6 than TPZ at even lower concentration. In addition, by introducing ataxia-telangiectasia-mutated (ATM) kinase inhibitors caffeine and KU-60019, we displayed that Q6-triggered apoptosis was attributed, at least partially, to DNA double-strand breaks generated by the topo II-targeting effect. Collectively, Q6 stood out for its better hypoxia-selectivity and topo II-poisoning than the parental compound TPZ. All these data shed light on the research of Q6 as a promising hypoxia-activated prodrug candidate for human hepatocellular carcinoma therapy.

Hypoxic microenvironment and hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is one of the most rapidly growing and prevalent cancers in the whole world. The characterized hypoxia region inside the HCC tumors has been recently found as the key driver of HCC malignance and treatment failure, leading to a variety of hypoxia-related biological consequences including angiogenesis, metastasis, metabolism deregulation and drug resistance, which ultimately resulted in treatment failure of HCC. This review will summarize the signaling pathways involved in hypoxia-mediated malignance of HCC and discuss current advances of hypoxia-targeted therapies.