PLC Cells Research Articles

PO-041 TNF pathway in metastatic colorectal cancer according to RAS status and implication of potassium channels

IntroductionDespite its role as a diagnostic and prognosis marker for Hepatocellular carcinoma (HCC), alpha fetoprotein (AFP) plays a key role in advancing tumorigenesis and tumour expansion. Telomerase, an enzyme elongating telomere length, is upregulated in 80% of cancers including HCC. Our team had elucidated a modulation of AFP by telomerase and protein kinase C (PKC). PKC family is formed of several isoforms, each with a specific cellular function and expression. The aim of this study is to investigate the interrelation between PKC isoforms, telomerase and AFP in HCC.Material and methodsPKC isoforms were quantified by RT-qPCR in two AFP secretory cell lines, HepG2/C3A and PLC/PRF/5 and two non-secretory AFP cell lines SNU-387 (hTERT+) and SKOV-3 (hTERT-). According to the results, the expression of four isoforms was suppressed by si-RNAs in HepG2/C3A and PLC cells. AFP and telomerase mRNA levels were quantified in transfected cells by q-PCR, and AFP secretion by ELISA. Toxicity and cell proliferation were assessed by WST-1. In order to examine the effect of the dual presence of AFP and hTERT on PKC isoforms, SNU-387 and SKOV-3 were transfected with AFP expression plasmid pCMV3-AFP, then PKC isoforms mRNA was assessed by qPCR.Results and discussionsFour PKC isoforms, alpha, beta, delta and epsilon exhibited the highest expression levels in all cells compared to the remaining isoforms. An increase in telomerase expression, AFP expression and secretion and cell proliferation reaching a maximum of two folds was observed after individual gene silencing of the four isoforms in HepG2/C3A cells; however, an inverse pattern was noticed when using PKC pan inhibitor Go6983. Similar results were observed in PLC cell line. The expression of the four isoforms increased in SNU-387 and SKOV-3 cells after 24 hour transfection. The effect persisted after 48 hour in SNU-387, contrary to SKOV-3 where the levels decreased returning to the controls expression levels.ConclusionTaken together, decreased individual PKC isoforms rise telomerase expression and AFP secretion. However, PKC isoforms overexpression requires the presence of hTERT in AFP secretory cells. Thus, these results show for the first time the possible inter relation linking PKC isoforms to both AFP and hTERT in HCC.

Curcumin suppress the growth of hepatocellular carcinoma via down-regulating SREBF1.

The aim of this study is to investigate the pharmacological effect of curcumin on hepatocellular carcinoma (HCC) Huh7 and PLC cells and to explore its mechanism in the pathological process by screening possible target genes. MTT assay was used to detect the effect of curcumin on the growth of Huh7 and PLC cells. The inhibitory effect was found to be in a time- and dose- dependent manner, and the cytoplasm was shrunk with round or elliptic morphology. Flow cytometry indicated that curcumin could significantly accelerate apoptosis of Huh7 and PLC cells, showing an obvious dosage effect. Retrieval in gene expression database and related gene-drug network analysis showed that the effect of curcumin on the apoptosis of HCC cells was related to SREBF1 (sterol regulatory element binding transcription factor 1) gene. In the in-vitro experiment, adding curcumin could significantly down-regulate SREBF1, and Western Blot suggested that down-regulation of SREBF1 could suppress the tumor growth. In conclusion, Curcumin could significantly suppress the tumor growth by way of down-regulating SREBF1 expression.

Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2 + dyshomeostasis in hepatocarcinoma PLC cells.

BackgroundNumerous experimental and epidemiological studies have demonstrated a link between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA) as well as hepatocellular carcinoma (HCC). The underlying molecular mechanism involved in the malignancy of CCA and HCC has not yet been addressed. Csseverin, a component of the excretory/secretory products of C. sinensis (CsESPs), was confirmed to cause obvious apoptotic inhibition in the human HCC cell line PLC. However, the antiapoptotic mechanism is unclear. In the present study, we investigated the cellular features of the antiapoptotic mechanism upon transfection of the Csseverin gene.MethodsIn the present study, we evaluated the effects of Csseverin gene overexpression on the apoptosis of PLC cells using an Annexin PE/7-AAD assay. Western blotting was applied to quantify the activation of caspase-3 and caspase-9, the mitochondrial translocation of Bax and the release of Cyt c upon Csseverin overexpression in PLC cells. Laser scanning confocal microscopy was used to analyze the changes of intracellular calcium. Fluorescence assay and immunofluorescence assays were performed to observe the changes of the mitochondrial permeability transition pore (MPTP).ResultsThe overexpression of Csseverin in PLC cells showed apoptosis resistance after the induction of apoptosis. Additionally, the activation of caspase-3 and caspase-9 was specifically weakened in Csseverin overexpression PLC cells. The overexpression of Csseverin reduced the increase in intracellular free Ca2+, thereby inhibiting MPTP opening in PLC cells. Moreover, Bax mitochondrial translocation and the subsequent release of Cyt c were downregulated in apoptotic Csseverin overexpression PLC cells.ConclusionsThe present findings suggest that Csseverin, a component of CsESPs, confers protection from human HCC cell apoptosis via the inactivation of membranous Ca2+ channels. Csseverin might be involved in the process of HCC through C. sinensis infestation in affected patients.

Inhibition of ERK signaling potentiates the anti-tumor activity of FL118 on hepatocellular carcinoma cells

Background: A novel camptothecin analogue, FL118, has been shown to exhibit superior anticancer activity over irinotecan on colon and lung cancer cells. It was reported that FL118 mainly performs its broadspectrum anticancer activity through inducing cancer cell apoptosis. Accumulated evidence indicated that ERK pathway was constitutively activated in liver cancer, thereby promoting tumorigenesis, development, metastasis and recurrence. In this study, we aimed to investigate the anticancer potency of FL118 on liver cancer cell lines and the effect of FL118 on extracellular regulated protein kinase (ERK) pathway. Methods: Viability of PLC and Huh7 cells was detected by MTT assay. Western blot assay was performed to assess phosphorylated and total ERK protein. And cell apoptosis was measured by Hochest-PI staining. Results: Our results revealed that FL118 can inhibit PLC and Huh7 cell viability and markedly induce their apoptosis. Western blot results indicated that ERK pathway was activated in huh7 and PLC cells after administration of FL118. In addition, the pro-apoptotic and antiviability ability of FL118 can be potentiated by ERK-specific inhibitor U0126. Conclusions: Our study confirmed good anticancer activity of FL118 on liver cancer, and targeting ERK pathway can significantly improve its anticancer potency.

Abstract 5190: Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation

Abstract Background: SET is a novel oncoprotein with growing evidence suggesting its roles in promoting the survival of hepatocellular carcinoma (HCC) and other types of malignant disease. Notably, pre-clinical data suggesting SET overexpression in cancer cell leaded to the development of resistance to various anti-cancer medications, and antagonizing SET reversely enhanced therapeutic efficacy. Radiotherapy (RT) is a commonly used anti-cancer modality, but its role for the treatment of HCC is largely limited for the concern of endogenous radioresistance. In this report, we explored the role of SET regarding the radiosensitivity of HCC cells. Furthermore, we explored whether SET serve as a valid target to enhance RT against HCC. Method: A panel of HCC cell lines, including Hep3B, PLC5, HA22T and HA59T, were used for in vitro experiments, and the PLC5 subcutaneous xenograft mice model was used for in vivo testing. HCC cells and tumors were treated with RT and/or SET antagonist and harvested for subsequent experiments. The viability of cancer cells were assessed by sub-G1, colony and sphere formation assay. Molecular events were determined by western blot and PP2A activity. Result: Through manipulating the expression of SET in cancer cells, we showed the crucial role of SET in mediating the effects of RT in HCC cells. Using colony formation and hepatosphere formation assay, we showed that RT significantly reduced the number and size of both tumor colony and sphere formed by Hep3B and PLC5 cells. In contrast, ectopic expression of SET in Hep3B and PLC5 cells abolished the effects of RT. More importantly, SET-knockdown oppositely potentiated the RT-induced growth inhibition in HCC cells. Based on our findings, we hypothesized that targeting SET may exert potential to enhance RT for the treatment of HCC. In order to validate our hypothesis, we tested the effects of RT in combine with a novel SET antagonist, EMQA, developed and characterized in our previous works. As expected, we found that EMQA significantly potentiated the anti-HCC effects of RT in vitro and in vivo. Moreover, we found that antagonizing SET to restore PP2A-mediated p-Akt downregulation was responsible for the synergism between EMQA and RT. Conclusion Oncoprotein SET plays a critical role in affecting the radiosensitivity of HCC cells. Using SET antagonist plus RT showed promising results in pre-clinical HCC models, and yields further investigation. Citation Format: Man-Hsin Hung, Chao-Yuan Huang, Chih-Ting Shih, Min-Hsien Tsai, Yung-Jen Hsiao, Tzu-I Chao, Feng-Shu Hsieh, Chung-Wai Shiau, Kuen-Feng Chen. Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2017-5190

Abstract 80: Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance

Abstract Background: Sorafenib is the only approved systemic agent for hepatocellular carcinoma (HCC). However, the efficacy of sorafenib is modest for HCC with low objective response rate and short time to tumor progression, indicating that the inherent and acquired resistance underlies the limited success of sorafenib in HCC. We conducted a pooled shRNA screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib. Materials and Methods: We introduced a pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatase and cytokine, into Huh7 HCC cells by retrovirus infection. The Huh7 cells after infection with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with vehicle or sorafenib at the dose of 30mg/kg/day for 4 weeks. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. Candidate genes were identified from those shRNAs with statistically different frequencies between vehicle-treated and sorafenib-treated xenografts, and were further validated by their impacts on sorafenib-sensitivity in HCC cells. Results: By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found that 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and a sorafenib-resistant subline of Huh7 cells (Huh7-SR), downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules such as XIAP and survivin in PLC5 and Huh7-SR cells. In 8 paired human HCC tissues, we found 3 of them have unequivocally increased expression of CDK5 in HCC cells of post-sorafenib HCC tissues compared with those of pre-treatment tissues. Conclusions: Our results suggest that increased CDK5 expression may mediate resistance to sorafenib in HCC in vivo, and targeting CDK5 is warranted further studies in the treatment of HCC. (This study was supported by grants MOST 103-2314-B-002 -181 -MY2, MOST 105-2314-B-002-194-, MOST 103-2314-B-002 -090 - & MOST 104-2314-B-002 -073). Citation Format: Tzu-Hsuan Lin, Wen-Chi Feng, Yu-Yun Shao, Li-Chun Lu, Ya-Jhen Chen, Ann-Lii Cheng, Chih-Hung Hsu. Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 80. doi:10.1158/1538-7445.AM2017-80

Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma

BackgroundLong-term infections by Clonorchis sinensis are associated with cholangitis, cholecystitis, liver fibrosis, cirrhosis, and even liver cancer. Molecules from the worm play vital roles in disease progress. In the present study, we identified and explored molecular characterization of C. sinensis granulin (CsGRN), a growth factor-like protein from C. sinensis excretory/secretory products (CsESPs).MethodsThe encoding sequence and conserved domains of CsGRN were identified and analysed by bioinformatics tools. Recombinant CsGRN (rCsGRN) protein was expressed in Escherichia coli BL21 (DE3). The localisation of CsGRN in adult worms and Balb/c mice infected with C. sinensis was investigated by immunofluorescence and immunohistochemistry, respectively. Stable CsGRN-overexpressed cell lines of hepatoma cells (PLC-GRN cells) and cholangiocarcinoma cells (RBE-GRN cells) were constructed by transfection of eukaryotic expression plasmid of pEGFP-C1-CsGRN. The effects on cell migration and invasion of CsGRN were assessed through the wound-healing assay and transwell assay. The levels of matrix metalloproteinase 2 and 9 (MMP2 and MMP9) in PLC-GRN or RBE-GRN cells were detected by real-time PCR (qRT-PCR). The levels of E-cadherin, vimentin, N-cadherin, zona occludens proteins (ZO-1), β-catenin, phosphorylated ERK (p-ERK) and phosphorylated AKT (p-AKT) were analysed by Western blotting.ResultsCsGRN, including the conserved GRN domains, was confirmed to be a member of the granulin family. CsGRN was identified as an ingredient of CsESPs. CsGRN was localised in the tegument and testes of the adult worm. Furthermore, it appeared in the cytoplasm of hepatocytes and biliary epithelium cells from infected Balb/c mouse. The enhancement of cell migration and invasion of PLC-GRN and RBE-GRN cells were observed. In addition, CsGRN upregulated the levels of vimentin, N-cadherin, β-catenin, MMP2 and MMP9, while it downregulated the level of ZO-1 in PLC-GRN/RBE-GRN cells. In total proteins of liver tissue from rCsGRN immunised Balb/c mice, vimentin level decreased, while E-cadherin level increased when compared with the control groups. Meanwhile, the levels of p-ERK reached a peak at 4 weeks post immunisation and the level of p-AKT did at 2 weeks after immunisation.ConclusionsThe encoding sequence and molecular characteristics of CsGRN were identified. As a member of granulin superfamily, CsGRN induced mesenchymal characteristics of PLC and RBE cells and was found to regulate the activities of the downstream molecules of the ERK and PI3K/AKT signalling pathways, which could contribute to the enhancement of cell migration and invasion.

Phenacyl group containing amide derivative of dehydroabietylamine exhibiting enhanced cytotoxic activity against PLC and MCF7 cancer cell lines

New amide derivatives of (+)-dehydroabietylamine, tricyclic abietane diterpene amine, were prepared using Zhongping’s protocol. (+)-N-(2-phenyl-acetyl)-dehydroabietylamine derivative (11) demonstrated noticeable growth attenuation of hepatocellular carcinoma cell lines including PLC/PRF/5, SNU475, Hep3B-TR, and Huh7 with IC50 of 7.4 µM, 9.8 µM, 11.7 µM, and 11.8 µM, respectively. A breast cancer cell line MCF7 was the most sensitive against amide 11 with lowest IC50 value (4.8 µM). Low cell confluence and increase in G2/M phase was recorded after 48 and 72 h of treatment of amide 11 on PLC/PRF/5 cell line. Finally, amide 11 has comparatively sufficient therapeutic role due to addition of N-phenacyl group at C-18. Amide 11 demonstrated as potential candidate for future cancer interference and research.

Combination of Photon and Carbon Ion Irradiation with Targeted Therapy Substances Temsirolimus and Gemcitabine in Hepatocellular Carcinoma Cell Lines.

This work investigates on putative cytotoxic effects in four different hepatocellular carcinoma (HCC) cell lines after irradiation with photons or carbon ions in combination with new targeted molecular therapy using either Temsirolimus (TEM) or Gemcitabine (GEM). The HCC cell lines HepG2, Hep3B, HuH7, and PLC were cultured and irradiated with photons or carbon ions at the Heidelberg Ion Beam Therapy Center using the raster-scanning method. For combination experiments, cell lines were first treated with Temsirolimus or GEM before irradiation. Cytotoxicity was measured by a clonogenic survival assay. The evaluation of the experiments and the obtained survival curves were based on the concept of additivity defined by Steel and Peckham. The results for the combination of carbon ions and both tested systemic substances TEM and GEM showed independent toxicities in all four cell lines. Supra-additive effects were observed in PLC cells for photon irradiation combined either with TEM or GEM and in HuH7 cells for the combination of photons with TEM. Addition of targeted therapy substances Temsirolimus and GEM to photon irradiation showed additive cytotoxicity in HCC cell lines, whereas independent toxicities where reached by the combination of carbon ions to these substances. It can be assumed that combining 12C with systemic substances only has independent effects because heavy ions cause direct damage because of their high-LET character resulting in complex and clustered double-strand breaks. Nonetheless, further investigations are warranted in order to determine whether addition of systemic therapy allows a reduction of radiation doses in combination therapy. This could possibly lead to better responses and tolerances in patients with HCC.

'Obligate' anaerobic Salmonella strain YB1 suppresses liver tumor growth and metastasis in nude mice.

The antitumor properties of bacteria have been demonstrated over the past decades. However, the efficacy is limited and unclear. Furthermore, systemic infection remains a serious concern in bacteria treatment. In this study, the effect of YB1, a rationally designed ‘obligate’ anaerobic Salmonella typhimurium strain, on liver tumor growth and metastasis in a nude mouse orthotopic liver tumor model was investigated. The orthotopic liver tumor model was established in nude mice using the hepatocellular carcinoma cell line MHCC-97L. Two weeks after orthotopic liver tumor implantation, YB1, SL7207 and saline were respectively administered through the tail vein of the mice. Longitudinal monitoring of tumor growth and metastasis was performed using Xenogen IVIS, and direct measurements of tumor volume were taken 3 weeks after treatment. In vitro, MHCC-97L and PLC cells were incubated with YB1 or SL7207 under anaerobic conditions. YB1 was observed to invade tumor cells and induce tumor cell apoptosis and death. The results revealed that all mice in the YB1 group were alive 3 weeks after YB1 injection while all mice in the SL7207 group died within 11 days of the SL7207 injection. The body weight decreased by ~9% on day 1 after YB1 injection and but subsequently recovered. Liver tumor growth and metastases were significantly inhibited following YB1 treatment. By contrast to the control group, a large number of Gr1-positive cells were detected on days 1 to 21 following YB1 treatment. Furthermore, YB1 also effectively invaded tumor cells and induced tumor cell apoptosis and death. In conclusion, YB1 suppressed liver tumor growth and metastasis in a nude mice liver tumor model. The potential mechanism may be through enhancing innate immune response and inducing tumor cell apoptosis and cell death.

Abstract 2831: Composite cyclin dependent kinase inhibition shows potent activity against hepatocellular carcinoma

Abstract Background: Almost a decade after its approval, sorafenib remains the only approved therapy for advanced hepatocellular carcinoma (HCC). Novel treatment is warranted for advanced HCC. Previous preclinical studies reported that inhibition of various cyclin dependent kinases (CDKs), whether cell cycle dependent or independent, showed activity against HCC. Dinaciclib is a potent inhibitor of CDK1, 2, 5, and 9 and has an acceptable safety profile in humans. We thus examined the efficacy of dinaciclib in HCC using preclinical models. Methods: In a panel of HCC cell lines with high Rb expression (HuH7, PLC5, HepG2, SK-Hep1, SNU387, SNU449, SNU426 and SNU475) and low Rb expression (Hep3B and HLE), dinaciclib was examined for its effect on cell viability. The impacts of dinaciclib on cell cycle distribution, apoptosis induction, and the expressions of phospho- (p-) Rb (the target of CDK1 and 2), p-ATM (the target of CDK5), c-myc, and p-RNA polymerase II (the target of CDK9) were explored in selected cell lines including HuH7 and PLC5. The in vivo efficacy of dinaciclib on HCC was tested in mouse xenografts of HuH7 cells implanted subcutaneously. Knockdown of various CDKs using RNA interference was conducted to explore the mechanisms of dinaciclib. Results: Dinaciclib showed potent anti-proliferative activities in all the tested HCC cell lines. The IC50s to dinaciclib by the MTT assay did not significantly differ between cell lines with high Rb expression (8.5 to 20.1 nM) and cell lines with low Rb expression (9.4 to 15.6 nM). After 48 hours of dinaciclib treatment, HuH7 and PLC5 cells showed G2/M arrest in a dose-dependent manner. Apoptosis assays including sub-G1 fraction analysis, DNA fragmentation detection, and cleaved PARP-1 confirmed the occurrence of apoptosis in HuH7 and PLC5 cells treated with dinaciclib. Dinaciclib at the concentration near the IC50 inhibited the phosphorylation of Rb, RNA polymerase II, and ATM in HuH7 and PLC5 cells; it also inhibited the expressions of multiple anti-apoptotic proteins including Mcl-1, XIAP, and survivin. In xenograft studies with HuH7 cells, mice receiving dinaciclib had a significantly slower tumor growth rate compared with mice receiving vehicles. The TUNEL assay showed that tumors treated with dinaciclib exhibited higher apoptosis activity. Mice tolerated the dinaciclib treatment well without significant body weight change. Knockdown of CDK 1, 2, 5, and 9 using RNA interference showed that CDK1 and CDK9 may contribute the most to the efficacy of dinaciclib. Conclusions: Composite knockdown of CDK 1, 2, 5, and 9 with dinaciclib showed potent activity against HCC. (This study was supported by Ministry of Science and Technology, Taiwan [MOST-103-2314-B-002-181-MY2, MOST-103-2314-B-002-090, MOST-103-2314-B-002-092]). Citation Format: Yi-Hsin Liang, Yu-Yun Shao, Yong-Shi Li, Hang Lin, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Composite cyclin dependent kinase inhibition shows potent activity against hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2831.

Apigenin inhibits NF-κB and snail signaling, EMT and metastasis in human hepatocellular carcinoma.

Apigenin is a naturally occurring compound with anti-inflammatory, antioxidant, and anticancer properties. In this study, we investigated the effects of apigenin on migration and metastasis in experimental human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo. Apigenin dose-dependently inhibited proliferation, migration, and invasion by PLC and Bel-7402 human HCC cells. It also suppressed tumor growth in PLC cell xenografts without altering body weight, thereby prolonging survival. Apigenin reduced Snai1 and NF-κB expression, reversed increases in epithelial-mesenchymal transition (EMT) marker levels, increased cellular adhesion, regulated actin polymerization and cell migration, and inhibited invasion and migration by HCC cells. Apigenin may therefore inhibit EMT by inhibiting the NF-κB/Snail pathway in human HCC.

Novel Aminopeptidase N Inhibitors with Improved Antitumor Activities

A series of aminopeptidase N (APN) inhibitors were designed and synthesized. Enzyme inhibitory, docking and antiproliferative studies were performed to evaluate the derived molecules. Molecule D15, with IC50 values of 10.9 μM, showed the best performance in the APN enzymatic inhibition assay. The binding pattern of molecule D9 and D15 in the active site of APN was predicted by docking studies. Hydrophobic and H-bond interactions were discovered to make key roles in the ligand-receptor bindings. Compared with the previous C7, several molecules such as D9, D14 and D15, exhibited significantly improved activities in inhibiting the growth of HL-60, ES-2, A549 and PLC cell lines. Keywords: Aminopeptidase N, inhibitor, structural modification, L-lysine based, docking.

Abstract 5421: HER3 inhibition has little efficacy on hepatocellular carcinoma cell lines

Abstract Background: HER3 is a member of the ErbB family of receptor tyrosine kinases that are involved in the pathogenesis of many malignancies, including hepatocellular carcinoma (HCC) (Hsieh et al: Hepatology 2011). When bound with neuregulins (NRGs) or in the presence of HER2 amplification, HER3 dimerizes with HER2 and activates the signaling transduction cascade. We planned this study to evaluate the efficacy of HER3 inhibition by an anti-HER3 monoclonal antibody, LJM716 (Novartis®), in HCC. Methods: Human HCC cell lines Huh7, HepG2, PLC5, Hep3B, and SK-Hep1 were used in the experiments. We used the quantitative RT-PCR to evaluate the mRNA expression levels of HER2, HER3 and NRGs in HCC cell lines. We used the MTT assay to evaluate the growth inhibitory efficacy of LJM716 alone or in combination with sorafenib, and used the Western blotting to study the expression phospho-HER3 downregulated by LJM716 in these cells. Results: None of the HCC cell lines have HER2 amplification. Huh7 and HepG2 had higher HER2 expression, SK-Hep1 had lower HER3 expression, SK-Hep1 and Hep3B had higher NRG1 and NRG2 expression, and PLC5 had higher NRG2 but not NRG1 expression compared with other HCC cells. In HCC cells cultured with ordinary serum-containing medium, we found LJM716 up to 20 μg/ml showed little suppressive effect in the proliferation of HCC cells; in HCC cells cultured in the presence of NRG1, LJM716 could induce a 20 to 30% growth-suppressive effect at best. LJM716 inhibited NRG1-induced phosphorylation of HER3 in HCC cells dose-dependently. The treatment of sorafenib slightly increased RNA expression levels of NRG1 and HER2 in PLC5, Huh-7, and HepG2 cells. We combined LJM716 and sorafenib to evaluate their combination effect in HCC cells, but found that LJM716 failed to show any synergistic effect to sorafenib. Conclusions: Despite inhibitory effect on HER3 phosphorylation, LJM716 showed minimal therapeutic efficacy on HCC cell lines, either alone or in combination with sorafenib. Citation Format: Yong-Shi Li, Yu-Yun Shao, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. HER3 inhibition has little efficacy on hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5421. doi:10.1158/1538-7445.AM2015-5421

Doubly end-on azido bridged mixed-valence cobalt trinuclear complex: Spectral study, VTM, inhibitory effect and antimycobacterial activity on human carcinoma and tuberculosis cells

Doubly end-on azido-bridged mixed-valence trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is afforded by employing a potential monoanionic tetradentate-N2O2 Schiff base precursor (2-[{[2-(dimethylamino)ethyl]imino}methyl]-6-methoxyphenol; HL). Single crystal X-ray structure reveals that in 1, the adjacent CoII and CoIII ions are linked by double end-on azido bridges and thus the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. Complex 1 is subjected on different spectral analysis such as IR, UV–vis, emission and EPR spectroscopy. On variable temperature magnetic study, we observe that during cooling, the χMT values decrease smoothly until 15K and then reaches to the value 1.56cm3Kmol−1 at 2K. Complex 1 inhibits the cell growth on human lung carcinoma (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human heptacellular (PLC5 cells) carcinoma cells. Complex 1 exhibits anti-mycobacterial activity and considerable efficacy on Mycobacterium tuberculosis H37Rv ATCC 27294 and H37Ra ATCC 25177 strains.

Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells.

Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines owing to ped/pea-15 expression level.

Omi/HtrA2 promotes cell apoptosis in human cancer cells. Early studies showed that primary hepatocellular carcinoma requires Omi/HtrA2 expression for cell apoptosis. Additionally, the Omi/HtrA2 pro-apoptotic marker demonstrated a difference in some cell types. However, how the Omi/HtrA2 pro-apoptotic marker reacts during the process of hepatocellular carcinoma cell apoptosis remains to be determined. Thus, we investigated the role and possible mechanism of Omi/HtrA2 on hepatocellular carcinoma cell apoptosis using various hepatocellular carcinoma cell lines. The results were analyzed using RT‑qPCR and western blot analysis. In the present study, we found that Omi/HtrA2 was overexpressed in hepatocellular carcinoma cell lines and induced hepatocellular carcinoma cell apoptosis. Additiionally, the only manner in which Omi/HtrA2 participated in cell death in PLC cells may be dependent on IAP-binding. Omi/HtrA2‑inducing HepG2 cell apoptosis may mainly depend on its serine protease activity while both IAP-binding and its serine protease activity participated in Hep3B cell apoptosis. This result suggested that Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines. PLC cells were also devoid of the expression of ped/pea-15 as the substrate of Omi/HtrA2 serine protease while ped/pea-15 was overexpressed in HepG2 and Hep3B cells and ped/pea-15 expression was higher in HepG2 cells than that in Hep3B cells. These results showed that Omi/HtrA2 overexpression promotes hepatocellular carcinoma cell apoptosis and the ped/pea-15 expression level causes this difference of the Omi/HtrA2 pro-apoptotic marker in the various hepatocellular carcinoma cell lines.

Erlotinib derivative inhibits hepatocellular carcinoma by targeting CIP2A to reactivate protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is a tumor suppressor, which is functionally defective in various cancers. Previously, we found that PP2A activity determined the anticancer effect of bortezomib and erlotinib in hepatocellular carcinoma (HCC) cells. Here, we tested a novel erlotinib derivative, TD52, in four HCC cell lines, PLC5, Huh-7, Hep3B and Sk-Hep1. Using MTT and flow cytometry, we showed that TD52 had more potent apoptotic effects than erlotinib in HCC cells. TD52-induced apoptosis was associated with dose- and time- dependent reactivation of PP2A and downregulation of cancerous inhibitor of protein phosphatase 2A (CIP2A) and p-Akt. Inhibition of PP2A or ectopic expression of CIP2A or Akt in PLC5 cells abolished the effects of TD52. Furthermore, we demonstrated that TD52 affected the binding of Elk-1 to the proximal promoter of the CIP2A gene, thus downregulating transcription of CIP2A. Importantly, TD52-induced tumor inhibition was associated with reactivation of PP2A and downregulation of CIP2A and p-Akt in vivo. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A determines the apoptotic effect induced by TD52. Our findings disclose the therapeutic mechanism of this novel targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.

Small interfering RNA (siRNA)-mediated knockdown of macrophage migration inhibitory factor (MIF) suppressed cyclin D1 expression and hepatocellular carcinoma cell proliferation.

Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.

RFX1-dependent activation of SHP-1 induces autophagy by a novel obatoclax derivative in hepatocellular carcinoma cells.

Obatoclax is a small molecule which targets the Bcl-2 family, and is to treat leukemia, lymphoma and lung carcinoma. Previously, an obatoclax analogue, SC-2001, was found to disrupt the protein-protein interactions of the Bcl-2 family and also repress Bcl-XL and Mcl-1 expression via STAT3 inactivation. Here, we report a novel mechanism of autophagy induction by SC-2001 in liver cancer cells. The findings indicate that SC-2001 induced the autophagy marker LC3-II in four hepatocellular carcinoma (HCC) cells. Autophagosomes induced by SC-2001-treated cells were confirmed by electron microscopy. SC-2001 activated SHP-1, dephosphorylated STAT3 and Mcl-1, and subsequently released free beclin 1. Overexpression of STAT3 and Mcl-1 in PLC5 cells attenuated the induction of SC-2001 on autophagy. Abolishment of SHP-1 by a specific inhibitor aboragated the autophagic effects induced by SC-2001. In addition, it was further revealed that RFX-1, a transcription factor of SHP-1, is a critical regulator in SC-2001-mediated autophagy. Downregulation of RFX-1 by si-RNA protected cells from SC-2001-induced autophagy. Importantly, Huh7 tumor-bearing nude mice treated with SC-2001 showed downregulation of Mcl-1 and p-STAT3 protein expression and upregulation of SHP-1, LC3II, and RFX-1 protein expression. In summary, our data suggest that SC-2001 induces autophagic cell death in a RFX1/SHP-1/STAT3/Mcl-1 signaling cascade.