Multidrug Resistance In Hepatocellular Carcinoma Research Articles

Abstract 5431: Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common type of cancer globally and is associated with a poor prognosis due to its high recurrence rate and resistance to treatment. Solid evidence showed the existence of cancer stem cells (CSCs) that are crucial for therapeutic resistance. However, there are only a few kinase inhibitors that have been found to specifically target liver T-ICs for the HCC treatment. To understand the regulatory mechanism of liver CSCs, we employed RNA-Seq analysis to compare the gene expression profiles, with a particular focus on protein kinases, between (1) sorted CD133+ liver CSCs and CD133- differentiated cells extracted from two HCC cell lines (Huh7 and PLC8024), as well as cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. Our analysis identified Serine/Threonine kinase 39 (STK39) as a commonly up-regulated protein kinase in both CD133+ liver CSCs and CSC-enriched hepatospheres. Based on our analysis of the TCGA data, a positive correlation has consistently been observed between STK39 mRNA expression and the expression of liver CSC markers, including CD24, CD133, and CD47. Utilizing publicly available transcriptome datasets from the NCBI Gene Expression Omnibus (GEO), including GSE14520 and GSE25097, we found that overexpression of STK39 is detected in HCC tumor tissues (p<0.001) and is significantly associated with poorer overall survival and disease-free survival. Through the use of lentiviral based overexpression and knockdown approaches, we have demonstrated the role of STK39 in regulating liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness, and the expression of liver CSC markers. STK39 expression was found to be significantly elevated in in HCC cells that were resistant to both sorafenib and lenvatinib, and its inhibition resulted in increased sensitivity of HCC cells to these treatments. To identify the downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. The analysis led to the identification of PARP1 as a novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, it was observed that the phosphorylation of PARP1 at Threonine 368 (T368) was significantly inhibited, indicating its role as a downstream effector of STK39-mediated liver CSC functions. Interestingly, STK39-mediated PARP1 phosphorylation led to development of resistance to PARP1 inhibitor, rucaparib. Additionally, STK39 plays a critical role in regulating PARP1-mediated chromatin decondensation, which enables it to open chromatin structure at the promoter regions of SOX2/OCT4. In summary, treatment of molecular targeted drugs including rucaparib and lenvatinib with STK39 inhibitor may be a potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5431.

New Insights into Traditional Chinese Medicine in Treatment of Diabetic Nephropathy

High-calorie diet and sedentary lifestyles are strongly associated with the development and onset of type 2 diabetes (T2D). According to the International Diabetes Federation, the global incidence of diabetes among individuals aged 20–79 years was 9.3% in 2019. The current estimated total number of patients with diabetes is 463 million, with an expected increase to over 578 million by 2030.[1] Systemic insulin resistance and insufficient insulin secretion in T2D result in several complications that severely impair a patients quality of life, especially the development of T2D nephropathy (T2DN).[2] Increasing evidence has shown that the dysregulation of TGF-β signaling contributes to the development of T2DN. For example, the TGF-β/Smad3 signaling pathway is dysregulated in the kidneys of T2D patients, where activation of Smad3 and loss of its inhibitor Smad7 lead to severe glomerular and tubulointerstitial fibrosis.[3] T2D associated with high levels of blood glucose and advanced glycation end-products can induce Smad3 phosphorylation in mesangial cells, tubular epithelial cells, and vascular smooth muscle cells in vitro.[3–4] Smad3 also reportedly downregulated the expression of the transcription factors Pdx-1, Nkx6.1, and NeuroD1 in the islet beta cells of db/db mice,[6,7] which transcriptionally suppresses the proliferation and function of islet beta cells and therefore further promotes the progression of T2D. In contrast, Smad7, a natural negative feedback regulator of Smad3, can reduce renal inflammation in diabetic nephropathy by promoting islet beta cell proliferation by targeting an NF-κB-dependent mechanism.[8] Thus, rebalancing the equilibrium between Smad3 and Smad7 in TGF-β downstream signaling may represent a therapeutic strategy for T2DN.[9] Traditional Chinese medicine (TCM) has been reported to have therapeutic effects in T2D. For example, a clinical study showed that Tianqi can significantly slow down diabetes progression and minimize diabetes risk by enhancing glucose tolerance.[10] Berberine, the main compound of Coptis, reduced insulin resistance in rats with streptozotocin-induced diabetes by suppressing oxidative stress and aldose reductase.[11] Combination therapy is a well-documented therapeutic protocol for TCM, which involves the simultaneous or sequential use of more than one herbal component to maximize the therapeutic outcome by parallel regulation of numerous signaling pathways and transcriptional events. In fact, our group has invented a novel TCM-based combination therapy named AANG, containing a natural Smad7 agonist, asiatic acid (AA) from Centella asiatica,[12] and the Smad3 inhibitor naringenin (NG) from Citrus or Millettiareticulata Benth,[13] which synergistically inhibit multidrug resistance in hepatocellular carcinoma by targeting Smad3-dependent p-glycoprotein expression at the transcriptional level.[14] These findings revealed the therapeutic potential of AANG in other human diseases associated with the aberration of TGF-β/Smad3 signaling. Encouragingly, our recently published article in the International Journal of Biological Sciences[15] provides novel insights into Smad3-dependent T2DN. In this study, we used AANG to rebalance the Smad3/Smad7 equilibrium in the diabetic injured kidneys of db/db mice, which not only reduced NF-κB-driven renal inflammation but also prevented Smad3-mediated renal fibrosis. Moreover, the collective results of the study found that AANG combination had a better effect than monotherapy with either AA or NG on the regulation of Smad3/Smad7 signaling in diabetic kidneys. Our data suggested that AANG effectively protected islet beta cells against diabetes-induced regression in a Smad3-dependent manner. Furthermore, we observed the synergistic effect of AANG on the induction of Smad7 for protecting the diabetic kidney against NF-κB-mediated renal inflammation.[16,17] In addition, we detected that AANG can inhibit the expression of the Smad3-dependent fibrogenic lncRNA Erbb4-IR[18] and inflammatory lncRNA LRNA9884[19] in db/db mice, which may also represent a protective mechanism against T2DN. Importantly, in the same study,[15] we unexpectedly discovered the preventive effect of AANG therapy on the primary disease T2D. We found that AANG synergistically prevented the onset of T2D in db/db mice, resulting in improved insulin sensitivity and glucose tolerance. The preventive effect of AANG on T2D was achieved by starting treatment in 4-week-old db/db mice at the pre-diabetic stage but not at the post-diabetic stage (12-week-old db/db mice). We further identified that AANG exerts a preventive effect on pancreatic islets, the niche of T2D onset. These interesting findings suggested that AANG can prevent T2D if applied to individuals at the pre-diabetic stage. In summary, our new study[15] demonstrated the efficiency of AANG in suppressing the development of T2DN in db/db mice by systematically rebalancing Smad3/Smad7 signaling in the pancreas and kidneys, which consequently suppressed Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. In addition, we unexpectedly discovered a novel therapeutic function of AANG in preventing T2D onset by reserving beta cell proliferation and insulin production capacity in islets under diabetic conditions. This research provides new insights and important clinical rationale for translating this AANG combined therapy as a unique and an effective treatment for T2DN, as well as a TCM-based formula for preventing T2D onset in patients with pre-diabetic conditions.

The inhibition of autophagy by spautin boosts the anticancer activity of fingolimod in multidrug-resistant hepatocellular carcinoma

The contribution of autophagy to drug resistance has been studied in several cancers. However, there is no clear evidence about the role of autophagy in the resistance to chemotherapy in cancers, such as hepatocellular carcinoma (HCC). HCC is characterized by a poor prognosis and limited therapeutic options. Moreover, the emergence of multidrug-resistance (MDR) hinders successful treatment. Therefore, understanding how autophagy is regulated in resistant HCC is essential for sensitizing this malignancy to chemotherapy. This work demonstrated that basal and induced autophagy differ between parental and resistant Hep3B cells. In optimum growth conditions, the basal level of autophagy was low in resistant Hep3B (Hep3B-R) cells compared to the wild-type Hep3B (Hep3B-P) cells. However, in metabolic or therapeutic stress conditions, the rate of autophagy flux was much faster in the resistant cells. The work also confirmed the pro-survival function of autophagy in HCC. Besides, it demonstrated that the autophagy inhibitor, spautin, acted synergistically with fingolimod (FTY720) to promote cell death. The combination treatment resulted in superior reactive oxygen species (ROS) production and significant induction of apoptosis. In addition, spautin potentiated the effect of FTY720 against cell survival pathways like the Akt and ERK. Interestingly, the results indicated that Hep3B-R cells were more sensitive to autophagy inhibition than their parental counterparts. Collectively, this work revealed that combining spautin with chemotherapeutic agents that induce cytoprotective autophagy such as FTY720 is a promising approach to overcome MDR in HCC.

Mitochondria-Targeting-Based of Paclitaxel-Loaded Triphenylphosphine-Pluronic F127-Hyaluronic Acid Nanomicelles in Multi-Drug Resistant Hepatocellular Carcinoma

Background:In this study a new novel nanomicelle (TPH) sco-loaded with triphenylphosphine (TPP)-Pluronic F127-hyaluronic acid (HA) and Paclitaxel (PTX) has been designed to treat multidrug resistant hepatocellular carcinoma (HCC).Methods:TPH was initially synthesized by ester bond formation with mitochondria-targeting TPP agent and TPH nanomicelles loaded with PTX (TPH/PTX) had outstanding physical characteristics in human multi drug-resistant HCC cell line Bel7402/5-FU. Cytotoxicity and hemocompatibility assessments, nanomicelle cellular absorption and mitochondrial targeting, andin vivoxenograft imaging was used to evaluate that the nonemicells delivered into target cell and components.Results:The results of fluorescence test showed that TPP could promote the fusion of nanomicells to human multi drugresistant HCC cell line Bel7402/5-FU, and targeted the mitochondria, and also improved the targeting and retention of drugs in liver tumors. The results of cell efficacy showed that TPH/PTX induced a strong apoptosis effect, which could significantly reduce the mitochondrial membrane Zeta potential, increase the level of intracellular ROS and the release of Caspase-3, significantly enhanced the pro-apoptotic protein (Bcl-2), decrease the expression level of anti-apoptotic protein (Bax).Conclusion:TPH/PTX has a promising mitochondrial targeting function, and can enhance the effect of drugs on promoting apoptosis of drug resistant HCC cells.

Synergistic antitumor efficacy of PD-1-conjugated PTX- and ZSQ-loaded nanoliposomes against multidrug-resistant liver cancers.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor chemotherapeutic efficiency due to multidrug resistance (MDR); it is very important to develop a targeted nanocarrier for the treatment of HCC. In this study, a programmed death ligand 1 (PD-L1)-conjugated nanoliposome was constructed for co-delivery of paclitaxel (PTX) and P-glycoprotein (P-gp) inhibitor zosuquidar (ZSQ) to overcome MDR in human HCC cells and tumors in vivo. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to examine the nanoparticles morphology and size; PD-1-conjugated PTX and ZSQ-loaded nanoliposomes (PD-PZLP) revealed a spherical shape with a size of 139.5 ± 10.7nm. Then, the physicochemical properties, as well as the drug loading capacity, release profile, cellular uptake, and cytotoxicity of the dual drug-encapsulated nanoliposomes were characterized. PD-PZLP displayed a high drug loading capacity of 20 ~ 30% for both PTX and ZSQ; the drug release of PTX and ZSQ in pH 5.0 was significantly faster than in pH 7.4. Cellular uptake study demonstrated PD-PZLP had higher internalization efficiency than non-targeted PZLP. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and reactive oxygen species (ROS) analysis demonstrated that PD-PZLP triggered an excessive ROS reaction and cell apoptosis compared with that of free PTX or ZSQ, which was also consistent with the cell antiproliferative effects in MTT assay. Furthermore, PD-PZLP could enhance synergistic antitumor effects on 7721/ADM xenograft tumor model, which also significantly alleviated hepatotoxicity as evident from the decreased aspartate transaminase (AST) and alanine transaminase (ALT) levels. Overall, PD-PZLP exhibited high loading capacity, significant synergistic effects, promising antitumor efficacy, and the lowest toxicity, which provide a promising strategy to overcome MDR in HCC.

Integrated Machine Learning and Bioinformatic Analyses Constructed a Novel Stemness-Related Classifier to Predict Prognosis and Immunotherapy Responses for Hepatocellular Carcinoma Patients.

Immunotherapy has made great progress in hepatocellular carcinoma (HCC), yet there is still a lack of biomarkers for predicting response to it. Cancer stem cells (CSCs) are the primary cause of the tumorigenesis, metastasis, and multi-drug resistance of HCC. This study aimed to propose a novel CSCs-related cluster of HCC to predict patients' response to immunotherapy. Based on RNA-seq datasets from The Cancer Genome Atlas (TCGA) and Progenitor Cell Biology Consortium (PCBC), one-class logistic regression (OCLR) algorithm was applied to compute the stemness index (mRNAsi) of HCC patients. Unsupervised consensus clustering was performed to categorize HCC patients into two stemness subtypes which further proved to be a predictor of tumor immune microenvironment (TIME) status, immunogenomic expressions and sensitivity to neoadjuvant therapies. Finally, four machine learning algorithms (LASSO, RF, SVM-RFE and XGboost) were applied to distinguish different stemness subtypes. Thus, a five-hub-gene based classifier was constructed in TCGA and ICGC HCC datasets to predict patients' stemness subtype in a more convenient and applicable way, and this novel stemness-based classification system could facilitate the prognostic prediction and guide clinical strategies of immunotherapy and targeted therapy in HCC.

An injectable thermosensitive hydrogel loaded with a theranostic nanoprobe for synergistic chemo-photothermal therapy for multidrug-resistant hepatocellular carcinoma.

Multi-drug resistance (MDR) is a complicated cellular defense mechanism for tumor cells to resist chemotherapy drugs, which is also the main cause of chemotherapy failure. In this study, a local injectable hydrogel delivery system was used to construct an on-demand sustained-release platform with the advantages of chemotherapy, photothermal therapy (PTT), and magnetic resonance imaging (MRI). It could achieve synergistic chemo-photothermal therapy and real-time evaluation of the therapeutic effects (via MRI) for MDR hepatocellular carcinoma (HCC). Furthermore, after a single administration, the prepared hydrogel with a theranostic nanoprobe could release the therapeutic agents on demand for up to 14 d. Firstly, doxorubicin (DOX) and gold-manganese oxide (Au-MnO) nanoparticles (NPs) were incorporated into liposome-based self-assembled micelles, then loaded into the thermosensitive hydrogel (F127) to form DOX@Au-MnO-L NPs/F127 hydrogel (DAML/H). The prepared NP complex showed a spherical morphology with a narrow size distribution. The prepared hydrogel drug delivery system had injectable properties and stable photothermal conversion. Both the DOX@Au-MnO-L NPs and DAML/H showed controlled drug release under near infrared (NIR) laser irradiation. The in vitro MRI studies indicated that the prepared DAML/H had a high relaxation rate (14.38 mM-1 s-1) and good MRI scanning sensitivity conditions. The in vitro and in vivo results suggested the synergistic chemo-photothermal therapy of DAML/H with NIR irradiation (808 nm, 1 W cm-2, 10 min) improved the antitumor efficacy for MDR HCC. The in vivo retention experiment of Au in tumors indicated that the prepared hydrogel drug delivery system (DAML/H) had a good ability to retain Au in the tumor for a long time (at least 14 d). The western blotting results revealed that DAML/H with laser treatment could effectively downregulate P-glycoprotein (P-gp), p53 and antiapoptotic protein (Bcl-2), whereas the expression level of proapoptotic protein (Bax) and caspase-3 were increased. Therefore, DAML/H could serve as a promising synergistic chemo-photothermal therapy for MDR HCC, and a single administration might achieve long-term (14 d), on-demand, sustained-release treatment of tumors.

CircRNA-001241 mediates sorafenib resistance of hepatocellular carcinoma cells by sponging miR-21-5p and regulating TIMP3 expression

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and its incidence is on the rise, closely related to advanced liver disease. Sorafenib chemotherapy is one of the main treatment options for patients with advanced HCC. Despite several reports on HCC multidrug resistance, the underlying regulatory mechanisms are still unclear. In this study, we found circ-001241 was significantly upregulated in HCC tissues and cells. Knockdown of circ-001241 markedly inhibited HCC cell proliferation and decreased sorafenib-resistance. More importantly, circRNA acts as a ceRNA to suppress the expression and activity of miR-21-5p, leading to the increase in TIMP3 expression. In addition, circRNA-001241 facilitated HCC sorafenib-resistance by regulating the miR-21-5p/TIMP3 axis. Taken together, our study elucidated the oncogenic role of circ-001241 in mediating sorafenib resistance in HCC, providing insights and opportunities to overcome sorafenib resistance in patients with advanced hepatocellular carcinoma.

Development of a DNA Aptamer against Multidrug-Resistant Hepatocellular Carcinoma for In Vivo Imaging.

Hepatocellular carcinoma (HCC) is a type of cancer that has high rates of recurrence and mortality. One of the most important factors that lead to treatment failure of HCC is the acquisition of multidrug resistance (MDR). Development of specific ligands for multidrug-resistant HCC will provide useful molecular tools for precise diagnosis and targeted theranostics. Herein, a multidrug-resistant HCC cell (HepG2/MDR)-specific aptamer was developed through Cell-SELEX (systematic evolution of ligands by exponential enrichment) technology. With dissociation constants lying in the nanomolar range, the molecularly designed PS-ZL-7c aptamer showed great selectivity to drug-resistant cancer cells. The in vivo imaging results illustrated that the PS-ZL-7c specifically accumulated in the drug-resistant tumors but not in drug-sensitive tumors and normal tissues, indicating that the PS-ZL-7c aptamer possessed excellent potential as a targeting ligand for precise diagnosis and target theranostics of multidrug-resistant HCC.

Lack of MOF Decreases Susceptibility to Hypoxia and Promotes Multidrug Resistance in Hepatocellular Carcinoma via HIF-1α

Hypoxia-inducible factor-1α (HIF-1α) promotes oncogenesis in hepatocellular carcinoma and is functionally linked to cell proliferation, chemoresistance, metastasis and angiogenesis. It has been confirmed that the low expression level of Males absent on the first (MOF) in hepatocellular carcinoma leads to poor prognosis of patients. However, potential regulatory mechanisms of MOF in response to hypoxia remain elusive. Our results demonstrate that MOF expression is negatively associated with HIF-1α expression in hepatocellular carcinoma tissues and in response to chloride-mimicked hypoxia in hepatocellular carcinoma cell lines. MOF regulates HIF-1α mRNA expression and also directly binds to HIF-1α to mediate HIF-1α N-terminal lysine acetylation, ubiquitination and degradation, with downstream effects on MDR1 levels. Functional inactivation of MOF enhances HIF-1α stability and causes cell tolerance to hypoxia that is insensitive to histone deacetylase inhibitor treatment. Dysfunction of MOF in hepatocellular carcinoma cells also results in chemoresistance to trichostatin A, sorafenib and 5-fluorouracil via HIF-1α. Our results suggest that MOF regulates hypoxia tolerance and drug resistance in hepatocellular carcinoma cells by modulating both HIF-1α mRNA expression and N-terminal acetylation of HIF-1α, providing molecular insight into MOF-dependent oncogenic function of hepatocellular carcinoma cells.

Insights into an Immunotherapeutic Approach to Combat Multidrug Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) has emerged as one of the most lethal cancers worldwide because of its high refractoriness and multi-drug resistance to existing chemotherapies, which leads to poor patient survival. Novel pharmacological strategies to tackle HCC are based on oral multi-kinase inhibitors like sorafenib; however, the clinical use of the drug is restricted due to the limited survival rate and significant side effects, suggesting the existence of a primary or/and acquired drug-resistance mechanism. Because of this hurdle, HCC patients are forced through incomplete therapy. Although multiple approaches have been employed in parallel to overcome multidrug resistance (MDR), the results are varying with insignificant outcomes. In the past decade, cancer immunotherapy has emerged as a breakthrough approach and has played a critical role in HCC treatment. The liver is the main immune organ of the lymphatic system. Researchers utilize immunotherapy because immune evasion is considered a major reason for rapid HCC progression. Moreover, the immune response can be augmented and sustained, thus preventing cancer relapse over the post-treatment period. In this review, we provide detailed insights into the immunotherapeutic approaches to combat MDR by focusing on HCC, together with challenges in clinical translation.

Phosphatidylinositol-3-kinase/Protein Kinase B Signaling Pathway: A Pivotal Factor for Stimulating Multidrug Resistance in Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a common malignant tumor. Chemotherapy is one of its principal modes of treatment, but multidrug resistance (MDR) poses an obstacle in HCC treatment. MDR is mainly mediated through drug transmembrane transporter activity, apoptosis inhibitory pathway abnormality, and changes in the intracellular enzyme. On top of that, phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway dysregulation is also one of the most common mechanisms of MDR. This signaling pathway plays critical roles in the differentiation, proliferation, and apoptosis of cancer cells, and is a new target of MDR treatment. This review article discusses the mechanisms of MDR in HCC with a strong emphasis on the significance and role of PI3K/Akt pathway in MDR, and the reversal of MDR by inhibiting PI3K/Akt pathway.

Asiaticoside Antagonizes Proliferation and Chemotherapeutic Drug Resistance in Hepatocellular Carcinoma (HCC) Cells.

BackgroundHepatocellular carcinoma (HCC) is the fifth most prevalent malignant tumor in China after lung cancer, gastric cancer, esophageal cancer, and breast cancer, and has a high mortality rate. Though there are a series of therapeutic strategies is now available for HCC in clinical practice, the 5-year survival rate after surgery is still low. In addition, multi-drug resistance (MDR) is one of the most important factors responsible for the low survival rate and poor therapy response in HCC. Hence, novel treatment strategies and molecules for HCC need to be developed.Material/MethodsWe assessed the effect of asiaticoside, a natural product derived from Centella asiatica (L.) Urban, on HCC cell proliferation and drug resistance.ResultsOur data indicated that asiaticoside significantly inhibited the proliferation of HCC cell lines QGY-7703 and Bel-7402 in a dose- and time-dependent manner. Moreover, asiaticoside significantly induced apoptosis in QGY-7703 and Bel-7402 cells. Treatment with asiaticoside also caused G1 cell cycle arrest in QGY-7703 and Bel-7402 cells. Western blot assay results indicated that the mechanism underlying the effects of asiaticoside involves inhibiting the activity of the PI3K/Akt and MAPK/ERK pathways. Furthermore, asiaticoside significantly antagonized P-gp-mediated MDR in HCC cells.ConclusionsOur results suggest that asiaticoside has the potential to be applied in the treatment of HCC patients, but further evidence is needed to confirm our results, particularly in vivo efficacy.

Digitoxin inhibits proliferation of multidrug-resistant HepG2 cells through G2/M cell cycle arrest and apoptosis.

Hepatocellular carcinoma (HCC) remains a challenge in the medical field due to its high malignancy and mortality rates particularly for HCC, which has developed multidrug resistance. Therefore, the identification of efficient chemotherapeutic drugs for multidrug resistant HCC has become an urgent issue. Natural products have always been of significance in drug discovery. In the present study, a cell-based method was used to screen a natural compound library, which consisted of 78 compounds, and the doxorubicin-resistant cancer cell line, HepG2/ADM, as screening tools. The findings of the present study led to the shortlisting of one of the compounds, digitoxin, which displayed an inhibitory effect on HepG2/ADM cells, with 50% inhibitory concentration values of 132.65±3.83, 52.29±6.26, and 9.13±3.67 nM for 24, 48, and 72 h, respectively. Immunofluorescence, western blotting and cell cycle analyses revealed that digitoxin induced G2/M cell cycle arrest via the serine/threonine-protein kinase ATR (ATR)-serine/threonine-protein kinase Chk2 (CHK2)-M-phase inducer phosphatase 3 (CDC25C) signaling pathway in HepG2/ADM cells, which may have resulted from a DNA double-stranded break. Digitoxin also induced mitochondrial apoptosis, which was characterized by changes in the interaction between Bcl-2 and Bax, the release of cytochrome c, as well as the activation of the caspase-3 and −9. To the best of our knowledge, the present study is the first report that digitoxin displays an anti-HCC effect on HepG2/ADM cells through G2/M cell cycle arrest, which was mediated by the ATR-CHK2-CDC25C signaling pathway and mitochondrial apoptosis. Therefore, digitoxin could be a promising chemotherapeutic agent for the treatment of patients with HCC.

Recent Advances in Asialoglycoprotein Receptor and Glycyrrhetinic Acid Receptor-Mediated and/or pH-Responsive Hepatocellular Carcinoma- Targeted Drug Delivery.

Hepatocellular carcinoma (HCC) seriously affects human health, especially, it easily develops multi-drug resistance (MDR) which results in treatment failure. There is an urgent need to develop highly effective and low-toxicity therapeutic agents to treat HCC and to overcome its MDR. Targeted drug delivery systems (DDS) for cancer therapy, including nanoparticles, lipids, micelles and liposomes, have been studied for decades. Recently, more attention has been paid to multifunctional DDS containing various ligands such as polymer moieties, targeting moieties, and acid-labile linkages. The polymer moieties such as poly(ethylene glycol) (PEG), chitosan (CTS), hyaluronic acid, pullulan, poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO) protect DDS from degradation. Asialoglycoprotein receptor (ASGPR) and glycyrrhetinic acid receptor (GAR) are most often used as the targeting moieties, which are overexpressed on hepatocytes. Acid-labile linkage, catering for the pH difference between tumor cells and normal tissue, has been utilized to release drugs at tumor tissue. This review provides a summary of the recent progress in ASGPR and GAR-mediated and/or pH-responsive HCC-targeted drug delivery. The multifunctional DDS may prolong systemic circulation, continuously release drugs, increase the accumulation of drugs at the targeted site, enhance the anticancer effect, and reduce side effects both in vitro and in vivo. But it is rarely used to investigate MDR of HCC; therefore, it needs to be further studied before going into clinical trials.

ABC Transporters: Regulation and Association with Multidrug Resistance in Hepatocellular Carcinoma and Colorectal Carcinoma.

For most cancers, the treatment of choice is still chemotherapy despite its severe adverse effects, systemic toxicity and limited efficacy due to the development of multidrug resistance (MDR). MDR leads to chemotherapy failure generally associated with a decrease in drug concentration inside cancer cells, frequently due to the overexpression of ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs), and breast cancer resistance protein (BCRP/ABCG2), which limits the efficacy of chemotherapeutic drugs. The aim of this review is to compile information about transcriptional and post-transcriptional regulation of ABC transporters and discuss their role in mediating MDR in cancer cells. This review also focuses on drug resistance by ABC efflux transporters in cancer cells, particularly hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) cells. Some aspects of the chemotherapy failure and future directions to overcome this problem are also discussed.

TGF-β1 elevates P-gp and BCRP in hepatocellular carcinoma through HOTAIR/miR-145 axis.

Multidrug resistance (MDR) is common in patients and has been linked to transforming growth factor-β1 (TGF-β1) and overexpression of drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), although the molecular mechanisms remain largely unknown. This study aimed to investigate the mechanisms underlying TGF-β1-induced MDR in hepatocellular carcinoma (HCC) cells. It was found that TGF-β1 upregulated HOX transcript antisense RNA (HOTAIR) expression in HCC cells. When drosophila mothers against decapentaplegic 4 (SMAD4) was silenced, HOTAIR expression was accordingly reduced. Meanwhile, miR-145 expression was increased in the case HOTAIR was silenced. If the enhancer of zeste homolog 2 (EZH2) was knocked down using small interfering RNA (siRNA), miR-145 expression was decreased. Then, the regulatory role of miR-145 in P-gp and BCRP expression was explored. The results showed that the expression of P-gp and BCRP protein was suppressed by miR-145 through binding to the 3'-untranslated regions (3'-UTRs) of P-gp and BCRP. In conclusion, our study revealed a novel mechanism explaining TGF-β1-induced MDR in HCC through upregulating P-gp and BCRP via the SMAD4/HOTAIR/miR-145 axis.

Role of microRNAs in the development of hepatocellular carcinoma and acquired drug resistance.

Hepatocellular Carcinoma (HCC) is a leading cause of cancer related death worldwide with a relatively poor survival rate. Aside from liver resection and subsequent transplant, the most effective and leading curative measure for HCC is the chemotherapeutic, sorafenib, a multi-kinase inhibitor used for treatment of late stage HCC. However, the effects of sorafenib are short lived due to the liver's rapid acquisition of Multi-Drug Resistance (MDR). MicroRNAs (miRNAs) have emerged as critical regulatory molecules for almost every biochemical pathway in an organism. The development and progression of HCC and acquired MDR are critically influenced by miRNAs through regulation of key genes in cell regulatory pathways. This review explores the involvement of miRNAs in development of HCC and their role in key signaling pathways for MDR in HCC.

Role of microRNAs in the development of hepatocellular carcinoma and drug resistance.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide and has a relatively poor survival rate. Aside from liver transplantation, the most effective and leading curative measure for HCC is the chemotherapeutic agent sorafenib, which is a multikinase inhibitor used for treating late-stage HCC. However, the effects of sorafenib are short-lived because of rapid acquisition of multi-drug resistance (MDR) by tumor cells. MicroRNAs (miRNAs) have emerged as crucial regulatory molecules for almost every biochemical pathway in humans. The progression of HCC and acquired MDR are critically influenced by miRNAs through regulation of key genes in cell regulatory pathways. This review explores the involvement of miRNAs in the development of HCC and their role in key signaling pathways leading to MDR in HCC.

Non-targeted metabolomic analysis on multidrug resistance hepatocellular carcinoma cell and reversal effect of annonaceous acetogenins

Multidrug-resistance (MDR) has been shown to play a critical role in the development of many diseases. In this study, we used metabolomic method to evaluate the MDR in hepatocellular carcinoma, and investigate regulatory of annonaceous acetogenins on MDR of hepatocellular carcinoma. Multivariate statistical analysis revealed that the MDR of SMMC 7721 together with changes in glutathione metabolism, arginine and proline metabolism, sphingolipid metabolism. Annonaceous acetogenins impact these metabolism pathways. Metabolic pathway analysis coupled with stoichiometry analysis can be an effective tool to understand MDR mechanism and to potentially find new MDR reversal agents.