Characterization of 9-nitrocamptothecin liposomes: anticancer properties and mechanisms on hepatocellular carcinoma in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. 9-Nitrocamptothecin (9NC) is a potent topoisomerase-I inhibitor with strong anticancer effect. To increase the solubility and stability, we synthesized a novel 9NC loaded liposomes (9NC-LP) via incorporating 9NC into liposomes. In the present study, we determined the effects of 9NC and 9NC-LP on in vitro and in vivo, and the underlying mechanisms.

Methodology/Principal Findings

We first analyzed the characteristics of 9NC-LP. Then we compared the effects of 9NC and 9NC-LP on the proliferation and apoptosis of HepG2, Bel-7402, Hep3B and L02 cells in vitro. We also investigated their anticancer properties in nude mice bearing HCC xenograft in vivo. 9NC-LP has a uniform size (around 190 nm) and zeta potential (∼−11 mV), and exhibited a steady sustained-release pattern profile in vitro. Both 9NC and 9NC-LP could cause cell cycle arrest and apoptosis in a dose-dependent and p53-dependent manner. However, this effect was not ubiquitous in all cell lines. Exposure to 9NC-LP led to increased expression of p53, p21, p27, Bax, caspase-3, caspase-8, caspase-9 and apoptosis-inducing factor, mitochondrion-associated 1 and decreased expression of Bcl-2, cyclin E, cyclin A, Cdk2 and cyclin D1. Furthermore, 9NC-LP exhibited a more potent antiproliferative effect and less side effects in vivo. Western blot analysis of the xenograft tumors in nude mice showed similar changes in protein expression in vivo.

Conclusions/Significance

In conclusion, 9NC and 9NC-LP can inhibit HCC growth via cell cycle arrest and induction of apoptosis. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo, which means it is a promising reagent for cancer therapy via intravenous administration.     

Recombinant Human Adenovirus-p53 Injection Induced Apoptosis in Hepatocellular Carcinoma Cell Lines Mediated by p53-Fbxw7 Pathway,Which Controls c-Myc and Cyclin E

F-box and WD repeat domain-containing 7 (Fbxw7/hAgo/hCdc4/Fbw7) is a p53-dependent tumor suppressor and leads to ubiquitination-mediated suppression of several oncoproteins including c-Myc, cyclin E, Notch, c-Jun and others. Our previous study has indicated that low expression of Fbxw7 was negatively correlated with c-Myc, cyclin E and mutant-p53 in hepatocellular carcinoma (HCC) tissues. But the role and mechanisms of Fbxw7 in HCC are still unknown. Here, we investigated the function of Fbxw7 in HCC cell lines and the anti-tumor activity of recombinant human adenovirus-p53 injection (rAd-p53, Gendicine) administration in vitro and in vivo. Fbxw7-specific siRNA enhanced expression of c-Myc and cyclin E proteins and increased proliferation in cell culture. rAd-p53 inhibited tumor cell growth with Fbxw7 upregulation and c-Myc and cyclin E downregulation in vitro and a murine HCC model. This effect could be partially reverted using Fbxw7-specific siRNA. Here, we suggest that the activation of Fbxw7 by adenoviral delivery of p53 leads to increased proteasomal degradation of c-Myc and cyclin E enabling growth arrest and apoptosis. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC.     

Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.     

Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

Sorafenib is the standard first-line drug for the treatment of advanced hepatocellular carcinoma (HCC), however, its therapeutic efficacy is not satisfactory due to primary or secondary resistance of HCC cells. In the present study, we identified Metaxin 1 (MTX1) as a new regulator of sorafenib resistance in HCC through genome-scale CRISPR activation (CRISPRa) screening. We found that MTX1 was frequently upregulated in HCC tissues and overexpression of MTX1 promoted HCC cell proliferation in vitro and in vivo. As well, MTX1 overexpression increased cell growth rate and decreased cell apoptosis upon sorafenib treatment. Consistently, the resistance induced by MTX1 was also observed in subcutaneous xenograft tumor model. Clinically, high expression of MTX1 was closely related with poor outcomes in HCC patients who received sorafenib treatment. Mechanistically, overexpression of MTX1 could promote HCC cell autophagy via interacting with and inhibiting CDGSH iron sulfur domain 1 (CISD1), an autophagy negative regulator. Taken together, our findings suggest that MTX1 is upregulated in HCC and contributes to sorafenib resistance via a possible mechanism involving CISD1 mediated autophagy.     

Indole-3-Carbinol Inhibits Nasopharyngeal Carcinoma Growth through Cell Cycle Arrest In Vivo and In Vitro

Nasopharyngeal carcinoma is a common malignant tumor in the head and neck. Because of frequent recurrence and distant metastasis which are the main causes of death, better treatment is needed. Indole-3-carbinol (I3C), a natural phytochemical found in the vegetables of the cruciferous family, shows anticancer effect through various signal pathways. I3C induces G1 arrest in NPC cell line with downregulation of cell cycle-related proteins, such as CDK4, CDK6, cyclin D1 and pRb. In vivo, nude mice receiving I3C protectively or therapeutically exhibited smaller tumors than control group after they were inoculated with nasopharyngeal carcinoma cells. The expression of CDK4, CDK6, cyclin D1 and pRb in preventive treatment group and drug treatment group both decreased compared with the control group. We conclude that I3C can inhibit the growth of NPC in vitro and in vivo by suppressing the expression of CDK and cyclin families. The drug was safe and had no toxic effects on normal tissues and organs.     

Microvesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Promote Human Renal Cancer Cell Growth and Aggressiveness through Induction of Hepatocyte Growth Factor

In our previous study, microvesicles (MVs) released from human Wharton''s jelly mesenchymal stem cells (hWJ-MSCs) retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC). By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0) was assessed. Cell counting kit-8 (CCK-8) assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro) or examination of cyclin D1 expression (in vivo) was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro) or MMP-2 and MMP-9 expression (in vivo). AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G_0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.     

Cell cycle regulation in the inner ear sensory epithelia: Role of cyclin D1 and cyclin-dependent kinase inhibitors

Sensory hair cells and supporting cells of the mammalian cochlea and vestibular (balance) organs exit the cell cycle during embryogenesis and do not proliferate thereafter. Here, we have studied the mechanisms underlying the maintenance of the postmitotic state and the proliferative capacity of these cells. We provide the first evidence of the role of cyclin D1 in cell cycle regulation in these cells. Cyclin D1 expression disappeared from embryonic hair cells as differentiation started. The expression was transiently upregulated in cochlear hair cells early postnatally, paralleling the spatiotemporal pattern of unscheduled cell cycle re-entry of cochlear hair cells from the p19^Ink4d/p21^Cip1 compound mutant mice. Cyclin D1 misexpression in vitro in neonatal vestibular HCs from these mutant mice triggered S-phase re-entry. Thus, cyclin D1 suppression is important for hair cell's quiescence, together with the maintained expression of cyclin-dependent kinase inhibitors. In contrast to hair cells, cyclin D1 expression was maintained in supporting cells when differentiation started. The expression continued during the neonatal period when supporting cells have been shown to re-enter the cell cycle upon stimulation with exogenous mitogens. Thereafter, the steep decline in supporting cell's proliferative activity paralleled with cyclin D1 downregulation. Thus, cyclin D1 critically contributes to the proliferative plasticity of supporting cells. These data suggest that targeted cyclin D1 induction in supporting cells might be an avenue for proliferative regeneration in the inner ear.     

Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody suppress hepatic cancer cells in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.

Methodology/Principal Findings

We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.

Conclusions

Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.     

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.     

Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.     

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.     

Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca²⁺ and dephosphorylated NFATc2 under low Ca²⁺ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.     

Concurrent versus Sequential Sorafenib Therapy in Combination with Radiation for Hepatocellular Carcinoma

Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G₁-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.     

7,3′,4′-Trihydroxyisoflavone Inhibits Epidermal Growth Factor-induced Proliferation and Transformation of JB6 P+ Mouse Epidermal Cells by Suppressing Cyclin-dependent Kinases and Phosphatidylinositol 3-Kinase

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3′,4′-Trihydroxyisoflavone (7,3′,4′-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3′,4′-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G₁ phase. As shown by Western blot, 7,3′,4′-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G₁ phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3′,4′-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3β/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3′,4′-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3′,4′-THIF provide a molecular basis for the possible development of new chemoprotective agents.