Apoptosis In Liver Cancer Cells Research Articles

Expression and purification of a recombinant ELRL-MAP30 with dual-targeting anti-tumor bioactivity

MAP30 (Momordica antiviral protein 30kD) is a single-chain Ⅰ-type ribosome inactivating protein with a variety of biological activities, including anti-tumor ability. It was reported that MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer. To determine whether adding two tumor targeting peptides could improve the antitumor activities of MAP30, we genetically modified MAP30 with an RGD motif and a EGFRi motif, which is a ligand with high affinity for αvβ3 integrins and with high affinity for EGFR. The recombinant protein ELRL-MAP30 (rELRL-MAP30) containing a GST-tag was expressed in E. coli. The rELRL-MAP30 was highly expressed in the soluble fraction after induction with 0.15 mM IPTG for 20 h at 16 °C. The purified rELRL-MAP30 appeared as a band on SDS–PAGE. It was identified by western blotting. Cytotoxicity of recombinant protein to HepG2, MDA-MB-231, HUVEC and MCF-7 cells was detected by MTT analysis. Half maximal inhibitory concentration (IC50) values were 54.64 μg/mL, 70.13 μg/mL, 146 μg/mL, 466.4 μg/mL, respectively. Proliferation inhibition assays indicated that rELRL-MAP30 could inhibit the growth of Human liver cancer cell HepG2 effectively. We found that rELRL-MAP30 significantly induced apoptosis in liver cancer cells, as evidenced by nuclear staining of DAPI. In addition, rELRL-MAP30 induced apoptosis in human liver cancer HepG2 cells by up-regulation of Bax as well as down-regulation of Bcl-2. Migration of cell line were markedly inhibited by rELRL-MAP30 in a dose-dependent manner compared to the recombinant MAP30 (rMAP30). In summary, the fusion protein displaying extremely potent cytotoxicity might be highly effective for tumor therapy.

AB4 inhibits Notch signaling and promotes cancer cell apoptosis in liver cancer.

The etiology for liver cancer has been clearly defined. Unfortunately, therapeutic approaches for liver cancer are rather limited, and liver cancer is insensitive to chemotherapy and radiotherapy. Traditional Chinese medicine (TCM) has become a promising strategy for cancer treatment as TCM elicits broad spectrum anticancer activity. In the present study, we evaluated the anticancer efficacy of AB4, an extract from the medical herb Pulsatilla chinensis (Bunge) Regel, in liver cancer in vitro and in vivo. We found that AB4 readily dose- and time-dependently inhibited liver cancer HepG2 and Huh-7 cell proliferation and colony formation. Western blot and flow cytometry analyses suggested that AB4 treatment induced liver cancer cell apoptosis. Moreover, these findings could be readily recaptured in vivo, in which the AB4 regimen resulted in tumor suppression and cancer cell apoptosis in xenograft tumor-bearing nude mice. Importantly, we noted that treatment with a Notch signaling inhibitor DAPT produced very similar anticancer efficacy in both HepG2 and Huh-7 cell lines, and administration of DAPT also efficiently suppressed HepG2 ×enograft outgrowth. To this end, we anticipated that AB4 and DAPT may act on the same signaling pathway, probably through inhibition of the Notch pathway. Indeed, we found decreased expression of Notch1 protein, as well as downstream targets Hes1 and Hey1, after AB4 treatment. Immunohistochemistry analysis further confirmed the suppression of Notch signaling in HepG2 ×enograft-bearing mice. Taken together, our study highlighted the anticancer efficacy of AB4 in liver cancer. We also provided preliminary data showing Notch as a therapeutic target of AB4. It would be interesting to investigate the anticancer efficacy of AB4 in other types of cancer with elevated Notch activity.

Protein kinase N1 promotes proliferation and invasion of liver cancer.

Protein kinase (PK) N1, also called PKC-related protein 1, participates in the proliferation, invasion and metastasis of various malignant tumors. However, the role of PKN1 in liver cancer remains to be elucidated. The present study investigated the expression of PKN1 using immunohistochemistry in surgical specimens from 36 patients and analyzed the correlation with VEGF, microvascular density (MVD), cell proliferation index (Ki67) and clinicopathological parameters. PKN1 was highly expressed in hepatocellular carcinoma (HCC) and was positively correlated with histological grading of HCC, Ki67 expression and MVD. PKN1 expression in moderately and poorly differentiated HCC was significantly higher compared with highly differentiated HCC. Expression of PKN1 was positively correlated with Ki67 and MVD, and Ki67 expression was positively correlated with MVD. The effects of PKN1 on proliferation, invasion and apoptosis of liver cancer cells were detected in vitro. Cell viability, migration and invasion were reduced and the apoptosis rate was significantly improved when PKN1 expression was silenced in liver cancer cells. Thus, PKN1 serves an important role in the development and progression of liver cancer. Inhibition of PKN1 activity may provide a promising therapeutic target for liver cancer.

γ‑irradiated prednisolone promotes apoptosis of liver cancer cells via activation of intrinsic apoptosis signaling pathway.

Prednisolone is an anti-inflammatory drug used to treat a number of conditions, including liver disease and cancer. Numerous studies have demonstrated that glucocorticoids such as prednisolone modified by ionizing radiation can promote anticancer activity in cancer cells. To the best of our knowledge, however, the effect of ionizing radiation on prednisolone structure and cancer cells has not yet been identified. The present study created a novel prednisolone derivative using γ-irradiation, and its anticancer properties were investigated in liver cancer cells. The present study confirmed the structure of the new prednisolone derivative using liquid chromatogram-mass spectrometry. MTT assays determined the cytotoxic effects of γ-irradiated (IR)-prednisolone in liver cancer cells. Flow cytometry analysis evaluated apoptosis, mitochondrial membrane potential and cell cycle distribution. Western blotting was used to analyze the proteins associated with apoptosis. The chromatogram profile revealed that IR-prednisolone produced a number of peaks compared with the single peak of the original prednisolone. In contrast to prednisolone, the MTT results showed that IR-prednisolone significantly prevented the growth of liver cancer cells. IR-prednisolone promoted apoptosis and arrested the cell cycle at the G0/G1 stage in Huh7 cells. IR-prednisolone also altered the mitochondrial membrane potential and activated caspase-associated proteins, which activated the intrinsic apoptotic signaling pathway. In conclusion, IR-prednisolone promoted anticancer effects in liver cancer cells via apoptosis activation. The present study demonstrated that IR-prednisolone may be a potential anticancer agent against liver cancer, although specific molecules have yet to be identified.

A Novel Circular RNA circCSPP1 Promotes Liver Cancer Progression by Sponging miR-1182.

IntroductionAberrant circular RNA (circRNA) expression has been extensively discovered for its involvement in both the initiation and progression of various cancers. Through screening circRNA profile, we identified a novel circRNA has_circ_0001806, which is termed as circCSPP1 in liver cancer. In the present study, we aim to investigate the role of circCSPP1 in the progression of liver cancer.MethodsFluorescence in situ hybridization (FISH) was used to detect the location of circCSPP1. Function studies including MTT, colony formation assay, transwell assay and flow cytometry were carried out to detect the malignant behaviour of circCSPP1 on liver cancer cells. Luciferase assay and RNA pull down were used to detect the interaction between miR-1182 and circCSPP1 as well as RAB15. Quantitative realtime (qPCR) and Western blot were performed to evaluate the RNA and protein expression, respectively.ResultsCircCSPP1 knockdown inhibited the proliferation, migration and invasion while promoted apoptosis of liver cancer cells. Mechanically, we predicted and verified the target miR of circCSPP1 which is miR-1182. miR-1182 was capable of reversing the effect of circCSPP1 on liver cancer cells. Moreover, miR-1182 was found to also target RAB15 to participate in the regulation of cell phenotype.DiscussionTaken together, circCSPP1 promoted progression of liver cancer cells via sponging miR-1182 which may serve as a novel prognostic and therapeutic target for liver cancer.

Reactive oxygen species (ROS)-response nanomedicine through knocking down a novel therapeutic target NEDD8-conjugating enzyme UBC12 (UBE2M) in the treatment of liver cancer

Knocking down the UBC12 with siRNA could significantly suppress the Neddylation pathway and result in an admirable anti-cancer effect. However, how to transport siRNA in vivo with low potential toxicity is an urgent problem to be solved. For enhancing bioavailability of siRNA, folic acid (FA)-modified PLGA (poly(lactic-co-glycolic acid))-TK (thioketal)-PEG (polyethylene glycol) nanomedicine was designed for siRNA delivery. The nano-vector possesses spherical morphology and admirable biocompatibility. After targeted endocytosis through the selective recognition between FA and its receptor in liver cancer cells, the genetic nanomedicine was triggered by high ROS level and released its cargo siRNA in tumor microenvironment. In vitro and In vivo results revealed that the genetic nanomedicine not only inhibits proliferation and promotes apoptosis of liver cancer cells, but also possesses excellent therapeutic efficacy by knowing down UBC12 and suppressing the Neddylation pathway as well as accumulating tumor-suppressive CRL substrates. Consequently, this work provides a novel approach to construct a biodegradable nanomedicine with targeted recognition and smart response. Moreover, it inhibits the viability of liver cancer cells with high efficiency and low potential toxicity. Importantly, the PLGA nanomedicine demonstrates the encouraging potential for multifunctional nano-system applicable for cancer therapy.

Study on the anti-hepatocarcinoma effect and molecular mechanism of Prunella vulgaris total flavonoids

Ethnopharmacological relevanceThe traditional use of Prunella vulgaris is for the treatment of liver cancer in a few areas of China. At present, it is used primarily for the treatment of thyroid cancer, throat cancer, and lymphosarcoma among others. However, there are few current scientific reports regarding its use for the treatment of liver cancer. In this paper, the effective treatment for liver cancer is studied to provide an experimental basis for the application of Prunella vulgaris, which is related to preparations in the treatment of liver cancer. Aim of the studyTo study the anti-hepatocarcinoma effect of Prunella vulgaris total flavonoids and explores the possible molecular mechanism. MethodsThe effects of Prunella vulgaris total flavonoids on the proliferation of SMMC-7721 cells were respected by RTCA analysis system. The tumor volume and weight were found in H22 tumor bearing mice. ELISA was used to observe the apoptosis and autophagy protein expressions in tumor tissue homogenate, along with the immune serum factor. Tumor tissue apoptosis was respected by the TUNEL method. And Bax, Bcl2, PI3K, Akt, mTOR, Beclin-1 and LC3-I/LC3-II expression were observed through Western blot. We also observed the expression of Beclin-1 and LC3-I/LC3-II through immunohistochemistry. ResultsThe total flavonoids of Prunella vulgaris inhibited the activity of SMMC-7721 cells, and reduced the tumor volume and weight in H22 tumor bearing mice. HE staining showed that the Prunella vulgaris total flavonoids inhibited liver metastasis of H22 tumor. The Prunella vulgaris total flavonoids significantly made the expressions of IL-6, TNF-α and IFN-γ immune factors increasing in the serum of tumor bearing mice, and the contents of caspase-3 and caspase-9 increase as well in tumor tissue homogenate. TUNEL showed that the mean density in the intervention group was significantly higher than that in the control group. P62 content in tumor tissue homogenate increased and ATG5 decreased after intervention. Immunohistochemistry showed Beclin-1 expression decreased and LC3-I/LC3-II increased in the tumor tissue. Western blot showed Bcl2, Beclin-1 expression decreased and Bax, PI3K, Akt, mTOR, LC3-I/LC3-II increased in the tumor tissue. ConclusionPrunella vulgaris total flavonoids have an obvious anti-hepatocarcinoma effect, and the mechanism may be linked to the inhibition of autophagy and promotion of apoptosis in liver cancer cells. The inhibition of autophagy may be related to activation of the PI3K/Akt/mTOR pathway.

Novel Copper(II) Complex with a 4-Acylpyrazolone Derivative and Coligand Induce Apoptosis in Liver Cancer Cells.

A novel pyrazolone-based copper complex [CuL(phen)(CH3OH)][CuL(phen)]·CH3CH2OH·CH3OH (P-FAH-Cu-phen) was synthesized and characterized. The asymmetric structural unit of P-FAH-Cu-phen was composed of two independent complex units [CuL(phen)(CH3OH)] and [CuL(phen)]:Cu12+ center with six coordination mode and Cu22+ center with five coordination mode. The growth of BEL-7404 cells and H22 cells was significantly inhibited by P-FAH-Cu-phen with IC50 values of 1.175 μg/mL and 1.097 μg/mL, respectively, which were much lower than IC50 of cisplatin for BEL-7404 cells (23.32 μg/mL) and H22 cells (27.5 μg/mL). P-FAH-Cu-phen induced cell cycle arrest at G2/M and apoptosis in BEL-7404 cells through mitochondria- and endoplasmic reticulum stress-associated pathways. Moreover, P-FAH-Cu-phen significantly suppressed the migration of BEL-7404 cells and the tumor growth in H22 tumor mouse model without severe side effects and improved the survival of tumor mice. The results suggested that P-FAH-Cu-phen might be a potential drug candidate for the treatment of live cancer.

ASPP2 expression predicts the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization.

Transcatheter arterial chemoembolization (TACE) induces ischemia-hypoxia and local chemotherapy-induced cytotoxicity which destroys cancerous cells. However, some patients do not respond to TACE. The causes for such a lack of response remain unclear. Recent studies have revealed that self-regulation of apoptosis-stimulating p53 protein 2 (ASPP2) may play an important role in promoting cell survival under hypoxic conditions as well as chemotherapy resistance via autophagy in various types of cancer. We measured the expression of ASPP2, autophagy-related proteins and apoptotic proteins by western blot assays. Multivariate logistic regression analysis was used to identify the independent risk factor. The present study found that ASPP2 expression was negatively correlated with that of BECN-1 (Beclin-1) in hepatocellular carcinoma (HCC) tissues. The expression of ASPP-1 was lower while that of Beclin-1 was higher in patients who underwent recurrence of HCC following TACE, than in those who do not undergo such a relapse. ASPP2 expression was also lower in cancerous tissues subjected to TACE, compared with that of directly resected cancerous tissue. The expression of LC3-II was also higher in patients with post-operative recurrence of HCC than in those without relapse. In vitro experiments showed that administration of an autophagy inhibitor, together with hypoxia activation and 5-FU treatment, promoted apoptosis in HepG2 liver cancer cells and primary HCC cells. Multivariate logistic regression analysis revealed that ASPP2 expression in cancer tissue following TACE is an independent risk factor for HCC recurrence as well as overall survival. Higher levels of ASPP2 expression were notably associated with higher objective responses evaluated via mRECIST. Thus, patients with resectable HCC showing high levels of ASPP2 expression may benefit from neoadjuvant TACE prior to resection. Our study provided a novel biomarker for HCC prognosis following TACE, based on cell survival mechanisms related to autophagy.

Research progress of neural precursor cells-expressed developmentally down-regulated protein-8 in liver diseases

Neural precursor cells-expressed developmentally down-regulated protein-8 (NEDD8) is one of the important members of the ubiquitin family, which plays an important role in maintaining cell stability, cell cycle regulation, signal transduction, transcription, and translation, DNA repair, and tumorigenesis through covalently bound substrates (also known as neddylation modification). In recent years, studies have found that the dysfunction of NEDD8 and its related enzymes is common in liver diseases, and is widely involved in the biological processes of hepatitis, liver fibrosis, proliferation, invasion, apoptosis and autophagy of liver cancer cells. This article focuses on the research progress of NEDD8 in liver diseases.

Kaempferol exhibits a synergistic effect with doxorubicin to inhibit proliferation, migration, and invasion of liver cancer

Kaempferol (KF), a flavonoid compound isolated from herbal medicines, has been reported to play a significant role in inhibiting certain types of cancer. Although recent studies reported that KF exerted inhibitive activity on liver cancer, they failed to elucidate the signaling pathways and synergistic effects in combination with chemotherapeutic drugs currently in use in the clinical setting. In the present study, the signaling pathways and synergistic effects of KF in liver cancer cells were investigated. Nine liver cancer cell lines were used to assess the inhibitive activity and synergistic effects of KF. Cellular behavioral experiments, such as viability, colony formation, cell cycle arrest, apoptotic, wound healing, and Transwell assays were used to assess the effects of KF on the proliferation, apoptosis, migration, and invasion of liver cancer cells. Western blotting was performed to validate the key signaling pathway elements underlying those cellular behaviors. KF exhibited inhibitory effects on nine liver cancer cell lines in time- and dose-dependent manners and was mostly nontoxic to the normal hepatocyte cells. The combination of KF and doxorubicin revealed a stronger inhibitive effect on the viability of liver cancer cells. Combination therapy also revealed higher suppressive effects on colony formation, cell cycle progression, survival, DNA damage response, and mitochondrial function. By western blotting assay, mitochondrial and caspase signaling pathways were determined to be involved in proliferation inhibition. In wound healing and Transwell invasion assays, combination therapy also exhibited more robust inhibitory activity in blocking the migration and invasion of liver cancer cells. PI3K/mTOR/MMP protein pathways were also revealed to be related to cell migration inhibition. KF alone exhibited an inhibitory effect on proliferation, migration, and invasion of liver cancer cells, and its synergistic effects revealed stronger inhibitory activities. The present data indicated that KF is a promising candidate as a complementary medicine to conventional chemotherapeutic drugs.

Demethylation of miR-34a upregulates expression of membrane palmitoylated proteins and promotes the apoptosis of liver cancer cells

BACKGROUNDLiver cancer is a common cancer and the main cause of cancer-related deaths worldwide. Liver cancer is the sixth most common cancer in the world. Although miR-34a and palmitoyl membrane palmitoylated protein (MPP2) are reportedly involved in various cell processes, their precise roles in liver cancer are still unclear.AIMTo investigate the expression of micro RNA 34a (miR-34a), methylation of the miR-34a promoter and the expression of MPP2 in liver cancer cells and their related mechanisms.METHODSTogether, 78 cases of liver cancer tissues and 78 cases of adjacent tissues were collected. The methylation degree of miR-34a promoter in liver cancer/ paracancerous tissue and liver cancer cells/normal liver cells, and the expression levels of miR-34a and MPP2 in the above samples were detected. Demethylation of liver cancer cells or transfection of liver cancer cells with miR-34a mimetic was performed. The MPP2 overexpression vector was used to transfect liver cancer cells, and the changes in proliferation, invasion, apoptosis, migration, and other biological functions of liver cancer cells after the above interventions were observed. Double luciferase reporter genes were used to detect the targeting relationship between miR-34a and MPP2.RESULTSClinical samples showed that the expression levels of miR-34a and MPP2 in liver cancer tissues were lower than those in the normal tissues. The methylation degree of miR-34a promoter region in liver cancer cells was higher than that in normal liver cells. After miR-34a demethylation/mimetic transfection/MPP2 overexpression, the apoptosis of liver cancer cells was increased; the proliferation, invasion and migration capabilities were decreased; the expression levels of caspase 3, caspase 9, E-cadherin, and B-cell lymphoma 2 (Bcl-2)-associated X protein were increased; and the expression levels of Bcl-2, N-cadherin, and β-catenin were decreased. Double luciferase reporter genes confirmed that MPP2 is targeted by miR-34a. Rescue experiments showed that small interfering MPP2 could counteract the promoting effect of miR-34a demethylation on apoptosis and the inhibitory effect on cell proliferation, invasion, and migration.CONCLUSIONmiR-34a demethylation upregulates the expression level of MPP2 in liver cancer cells and promotes the apoptosis of liver cancer cells. miR-34a demethylation is a potential method for liver cancer treatment.

Zinc oxide nanoparticles promotes liver cancer cell apoptosis through inducing autophagy and promoting p53.

Emerging evidence has highlighted the promising potential of the application of Zinc Oxide nanoparticles (nano-ZnO) but the mechanism by how it functions in liver cancer remains elusive. We aimed to explore the effect of nano-ZnO on liver cancer cells. Liver cancer cells Huh7 cells were transfected with GFP-LC3, and then, treated with DMSO, Sorafenib, and nano-ZnO respectively to set blank group, Sorafenib control group, and nano-ZnO group followed by the analysis of the expression of GFP-LC3, p53, and Caspase by Western blot and RT-qPCR, cell apoptosis and viability by flow cytometry and CCK-8 assay. With a diameter of nano-ZnO 14.13±0.92 nm, the amount of GFP-LC3 protein was increased after treatment of nano-ZnO. Besides, the expressions of GFP-LC3, p53, and Caspase in Sorafenib group and nano-ZnO group were significantly higher than that of control group, while their levels were highest in nano-ZnO group (p<0.05). In nano-ZnO group, the values of D450nm at 24 h, 48h, and 72 h were 0.56±0.06, 0.39±0.05, and 0.22±0.04, respectively, and the apoptotic rate (83.11±2.79%) was significantly lower than that of blank group and control group. Nano-ZnO induced autophagy, upregulated the p53 gene, and facilitated the apoptosis of liver cancer cells, indicating that nano-ZnO might be a therapeutic approach for the treatment of liver cancer patients.

Irigenin exerts anticancer effects on human liver cancer cells via induction of mitochondrial apoptosis and cell cycle arrest

Irigenin has been reported to exhibit remarkable anticancer effects against several human cancers. Nonetheless, the anticancer effects of irigenin against the human liver cancer cells are still largely unexplored. Consistently, this study was designed to evaluate the anticancer effects of irigenin against human liver cancer cells and to unveil the underlying molecular mechanisms. The results showed that irigenin significantly (p < 0.05) inhibited the growth of the human HepG2 and SNU-182 liver cancer cells with an IC50 value of 14 µM. Nonetheless, the cytotoxic effects of irigenin against the normal THLE-2 cells were comparatively lower as evident from the IC50 of 120 μM. The AO/EB and DAPI staining showed that irigenin induces apoptosis in the human liver cancer cells. Annexin V/PI staining assay revealed a significant (p < 0.05) increase in the percentage of apoptotic HepG2 and SNU-182 liver cancer cells upon treatment with irigenin. It was found that the number of apoptotic HepG2 and SNU-182 cells enhanced from 2.3 to 41.75% and 1.16 to 51.9% at IC50, respectively. Western blot showed a considerable increase in Bax and decrease in the Bcl-2 expression upon irigenin treatment further confirming the induction of apoptosis. Flow cytometric analysis revealed that irigenin also induces G2/M cell cycle arrest of HepG2 and SNU-182 cells. The percentage of G2/M phase HepG2 and SNU-182 cells increased from 17.92 to 34.35% and 23.97 to 38.23% at IC50, respectively This was also accompanied by decrease in the expression of CDK1 and Cyclin-B in HepG2 and SNU-182 cells. Taken together, the results of the present study suggest that irigenin inhibits the growth of the human liver cancer cells via induction of apoptosis and cell cycle arrest. These results point towards the potential of irigenin as a lead for the development of chemotherapy for liver cancer.

6-Shogaol Suppresses the Progression of Liver Cancer via the Inactivation of Wnt/[Formula: see text]-Catenin Signaling by Regulating TLR4.

Liver cancer is a gastrointestinal malignant tumor with high lethality. The prognosis of liver cancer remains poor. Compounds derived from natural products have been confirmed to alleviate the progression of various diseases, including cancers. Additionally, 6-Shogaol has been reported to induce apoptosis in liver cancer cells. However, the mechanism by which 6-shogaol regulates apoptosis in liver cancer cells remains unclear. To investigate the function of 6-shogaol in liver cancer, RT-qPCR and western blotting were used to detect the expression of TLR4 and FOXO3a in liver cancer cells, respectively. The OD value of liver cancer cells was measured using the MTT assay. Flow cytometry was used to measure cell apoptosis. 6-Shogaol inhibited the growth of liver cancer cells. TLR4 and Wnt/[Formula: see text]-catenin were upregulated in liver cancer cells, and FOXO3a was inactivated, but 6-Shogaol reversed the expression of TLR4, Wnt/[Formula: see text]-catenin and FOXO3a in liver cancer cells. Additionally, TLR4 overexpression partially reversed the inhibitory effect of 6-shogaol on the progression of liver cancer cells via Wnt/[Formula: see text]-catenin signaling. Furthermore, the 6-shogaol-induced increase in FOXO3a expression in liver cancer was notably suppressed by TLR4 or Wnt/[Formula: see text]-catenin upregulation. Thus, 6-Shogaol suppresses the progression of liver cancer by mediating Wnt/[Formula: see text]-catenin signaling and is a potential agent for the treatment of liver cancer.

Study of RNA38 reverses apatinib resistance in hepatocellular carcinoma via regulating NTNG1

Presenter: Xing Gao | Guangxi Medical University Background: Apatinib is a small-molecule anti-angiogenesis drug, shows favorable efficacy in advanced liver cancer. However, drug resistance hinders the further survival and benefit of patients. RBM38 is a RNA-binding protein, binds to RNA for the post-transcriptional regulation. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to analyze the expression levels of RBM38 and NTNG1in hepatocellular carcinoma cells. The role of RBM38 and NTNG1 in hepatocellular carcinoma (HCC) cells drug resistance were further detected by Cell counting kit-8 (CCK-8) assays. Cell cycle and cell proliferation were analyzed by flow cytometry. Cell migration and invasion were detected using a transwell assay. The expression of the related drug-resistant proteins were assessed by western blot. The xenograft model was established to examine the potential role of RBM38 reverses apatinib resistance in HCC. Results: Our study found that the RBM38 is closely related to the sensitivity of HCC to apatinib. After apatinib stimulation, the level of RBM38 was increased in liver cancer cells. While the expression of RBM38 was inhibited in apatinib-resistant liver cancer cells. The results of in vitro and in vivo experiments demonstrated that overexpression of the RBM38 gene reverses the apatinib resistance of liver cancer cells, the expression of ABCG2 and other related drug-resistant proteins also decreased. Overexpression of RBM38 inhibits proliferation, invasion, migration and induces apoptosis of liver cancer cells, and suppresses xenograft tumor formation. In the present study, high-throughput gene chip screening revealed that NTNG1 is the most significant differentially expressed genes following RBM38 gene overexpression. After apatinib stimulation, the level of NTNG1 was decreased in liver cancer cells. While the expression of NTNG1 was increased in apatinib-resistant liver cancer cells. After NTNG1 was further overexpressed in the apatinib-resistant liver cancer cell model overexpressed by RBM38, in vitro and in vivo experiments confirmed that the sensitivity of liver cancer cells to apatinib was reduced, and the expression of ABCG2 and other related drug-resistant proteins also increased. Overexpression of NTNG1 promotes inhibits proliferation, invasion, migration, and inhibits apoptosis of liver cancer cells, and enhances xenograft tumor formation. Conclusion: This study preliminarily clarified the biological effect and molecular mechanism of RBM38 reverses apatinib resistance in hepatocellular carcinoma via regulating NTNG1, providing a new theoretical basis and experimental basis for the drug resistance mechanism of advanced liver cancer targeted therapy.

Dichloroacetate enhances the antitumor effect of pirarubicin via regulating the ROS-JNK signaling pathway in liver cancer cells.

Aim: Liver cancer is one of the most common malignancies and has a high recurrence rate. However, current treatment strategies do not achieve satisfactory outcomes in the clinic. To explore a new strategy to enhance the effectiveness of chemotherapy in liver cancer, we investigated whether dichloroacetate (DCA) could enhance the sensitivity of liver cancer cells to pirarubicin (THP).Methods: Liver cancer cells were treated with DCA alone, THP alone, or DCA and THP combined. Cell viability was determined by the CCK-8 assay. Cell apoptosis was analyzed by flow cytometer. Reactive oxygen species (ROS) were detected using a CM-H2DCFDA fluorescence probe. Protein levels were identified by immunoblotting.Results: The results revealed that DCA significantly enhanced the antitumor effect of THP in liver cancer cells. Changes in morphology and adherence ability were observed, as well as decreased cell viability. The results of flow cytometry showed that the combination of THP and DCA significantly increased apoptosis of liver cancer cells. Moreover, compared with THP alone, combination treatment with DCA significantly increased THP-triggered ROS generation in liver cancer cells. The antioxidant N-acetyl-L-cysteine reversed the synergistic effect of DCA and THP on ROS generation, cell viability and apoptosis. Furthermore, phosphorylation of c-Jun N-terminal kinase (JNK) was significantly increased in the DCA and THP combination group. The effects of DCA and THP on cell viability and apoptosis were inhibited by the JNK inhibitor SP600125.Conclusion: The results obtained in the present study indicated that DCA enhanced the antitumor effect of THP in liver cancer cells via regulating the ROS-JNK signaling pathway.

Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently.Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety.Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs.Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

The effect of propofol on the proliferation and apoptosis of hepatocellular carcinoma cells through TGF-Β1/Smad2 signaling pathway

ABSTRACT Malignant tumors are a serious threat to human health. Surgical resection is the most effective treatment for liver cancer. However, liver cancer is mostly found at an advanced stage, is difficult to remove by surgery, and has a very high recurrence rate after surgery. The current liver cancer treatment drugs have serious side effects, and the treatment effect is not ideal, far from meeting the clinical needs. Based on this, this paper studies the effect of propofol on the proliferation and apoptosis of liver cancer cells through the TGF-B1/Smad2 signaling pathway, and explores the proliferation, adhesion and apoptosis of cancer cells in patients with propofol. This paper uses a comparative experiment. With medical imaging method, 80 rats with liver cancer in the same period were cultured. High-precision microscope and radiolocation method were used to observe and record the whole process of propofol regulating Smad2 signal pathway. The results show that propofol can effectively inhibit the proliferation of cancer cells in patients with liver cancer. Propofol can increase the activity and content of transforming growth factor-β1 by 12% and 20%, respectively, and then inhibit the proliferation rate of liver cancer cells by 10% through the Smad2 signaling pathway, and exponentially increase the apoptotic number of liver cancer cells. This shows that propofol has a significant inhibitory effect on the cycle of liver cancer cells. Under the action of propofol, the life cycle of liver cancer cells is shortened, which provides a certain theoretical basis for the treatment of liver cancer.

MicroRNA-638 induces apoptosis and autophagy in human liver cancer cells by targeting enhancer of zeste homolog 2 (EZH2)

Liver cancer is of the devastating human cancers and its incidence is increasing at an alarming rate. The clinical outcomes are far from descent due to lack of efficient therapeutic targets and chemotherapeutic agents. Studies have revealed the therapeutic implications of microRNAs in the management of different human cancers. This study was designed to explore the role and therapeutic potential of miR-638 in liver cancer via modulation of zeste homolog 2 (EZH2). The results revealed significant (P < 0.05) downregulation of miR-638 in human liver cancer tissues and cell lines. Overexpression of miR-638 led to a significant (P < 0.05) decline in liver cancer cell proliferation. Nonetheless, inhibition of miR-638 could promote the proliferation of the human liver cancer cells. The DAPI and annexin V/PI staining assays revealed that miR-638 induces apoptosis in human liver cancer cells which was accompanied by enhancement of Bax and depletion of Bcl-2 expression. Furthermore, miR-638 overexpression also leads to a significant (P < 0.05) increase of autophagosomes and autolysosomes in liver cancer cells suggestive of autophagy. The induction of autophagy was further confirmed by increase and decrease in expression of LC3B-II and Beclin-1 proteins, respectively. In contrary, inhibition of miR-638 prevented both apoptosis and autophagy of the liver cancer cells. In silico analysis and the dual luciferase assay revealed EZH2 as the molecular target of miR-638 at post-transcriptional level. The qRT-PCR showed that EZH2 to be significantly (P < 0.05) upregulated in the human liver cancer tissues and cell lines. However, the expression of EZH2 was considerably suppressed upon miR-638 overexpression in SNU-423 cells. Taken together, these findings suggest the tumor-suppressive role of miR-638/EZH2 axis liver cancer and point towards the potential of miR-638 as therapeutic target in the treatment of liver cancer.