Novel affibody molecules as potential agents in molecular imaging for MAGE-A3-positive tumor diagnosis.

BACKGROUND: The cancer-testis protein melanoma antigen A3 (MAGE-A3) is highly expressed in a broad range of malignant tumor forms. It has been confirmed that affibody molecules, a novel family of small (∼6.5 kDa) targeting proteins, are useful agents for molecular imaging and targeted tumor treatment. As a novel agent for in vivo molecular imaging detection of MAGE-A3-positive tumors, the efficacy of affibody molecules was assessed in this research. METHODS: In this study, three cycles of phage display library screening resulted in the isolation of two new affibody molecules (ZMAGE-A3:172 and ZMAGE-A3:770) that attach to MAGE-A3. These molecules were then expressed in bacteria and purified. The affibody molecules with high affinity and specificity were evaluated using western blotting, immunohistochemistry, indirect immunofluorescence, surface plasmon resonance, and near-infrared optical imaging of tumor-bearing nude mice. RESULTS: The selected ZMAGE-A3 affibodies can precisely bind to the MAGE-A3 protein in living cells and display high-affinity binding to the MAGE-A3 protein at the molecular level. Furthermore, the accumulation of DyLight755-labeled ZMAGE-A3:172 or ZMAGE-A3:770 in MAGE-A3-positive tumors was achieved as early as 30 min and disappeared at 48 h post-injection. CONCLUSION: Our findings support the potential of the two MAGE-A3 protein-binding affibody molecules for their use as molecular imaging agents.

Novel Bifunctional Affibody Molecules with Specific Binding to Both EBV LMP1 and LMP2 for Targeted Therapy of Nasopharyngeal Carcinoma.

Antibodies are considered highly specific therapeutic agents in cancer medicines, and numerous formats have been developed. Among them, bispecific antibodies (BsAbs) have gained a lot of attention as a next-generation strategy for cancer therapy. However, poor tumor penetration is a major challenge because of their large size and thus contributes to suboptimal responses within cancer cells. On the other hand, affibody molecules are a new class of engineered affinity proteins and have achieved several promising results with their applications in molecular imaging diagnostics and targeted tumor therapy. In this study, an alternative format for bispecific molecules was constructed and investigated, named ZLMP110-277 and ZLMP277-110, that targets Epstein-Barr virus latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2). Surface plasmon resonance (SPR), indirect immunofluorescence assay, co-immunoprecipitation, and near-infrared (NIR) imaging clearly demonstrated that ZLMP110-277 and ZLMP277-110 have good binding affinity and specificity for both LMP1 and LMP2 in vitro and in vivo. Moreover, ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, significantly reduced the cell viability of C666-1 and CNE-2Z as compared to their monospecific counterparts. ZLMP110-277 and ZLMP277-110 could inhibit phosphorylation of proteins modulated by the MEK/ERK/p90RSK signaling pathway, ultimately leading to suppression of oncogene nuclear translocations. Furthermore, ZLMP110-277 and ZLMP277-110 showed significant antitumor efficacy in nasopharyngeal carcinoma-bearing nude mice. Overall, our results demonstrated that ZLMP110-277 and ZLMP277-110, especially ZLMP277-110, are promising novel prognostic indicators for molecular imaging and targeted tumor therapy of EBV-associated nasopharyngeal carcinoma.

NSUN2 Promotes Tumor Progression and Regulates Immune Infiltration in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in the head and neck with a complex etiology, such as environmental factors, genetic factors, and Epstein-Barr virus infection. The NOP2/Sun domain family, member 2 (NSUN2) is a methyltransferase of m5C methylation modification that has been reported to be involved in the occurrence and progression of various tumors, but its role in NPC remains unclear. In this study, we found that NSUN2 was upregulated in NPC and predicted a poor prognosis for NPC patients in both GEO datasets and our tissue microarrays containing 125 NPC tissues. Next, we demonstrated that NSUN2 promoted the proliferation, migration, and invasion of NPC cells in vitro. Additionally, the differential expression genes between NSUN2-high and low expression patients were mainly enriched in multi-immune cell activation and proliferation. Furthermore, NSUN2 negatively regulates immune cell infiltration in the tumor microenvironment (TME) of NPC, which indicates that the NSUN2 level may be negatively correlated with the sensitivity of immunotherapy and chemotherapy. In conclusion, our findings highlight that NSUN2 might act as an important oncogene involved in NPC progression and serve as a potential biomarker to predict poor prognosis and drug sensitivity of NPC patients.

Sustained expression of HPV16 E7 oncoprotein promotes p-AKT(Ser473)/p-Src(Tyr527) signaling to drive precancerous lesions to invasive cervical cancer.

Human papillomavirus (HPV) E7 oncogene plays the most important role in cervical cancer. However, whether E7 oncoprotein is continuously expressed, associated with AKT(Ser473)/p-Src(Tyr527) signaling to trigger cervical carcinogenesis remains unclear. Here, we explored first if HPV16 E7 oncoprotein could be detected in clinical biopsies and is sustainedly expressed, and then investigated how this oncoprotein interacted with AKT(Ser473)/p-Src(Tyr527) signaling in cancer progression. We used ZHPV16E7384 affibody to detect E7 expression in HPV16-positive cervical cancer biopsies and animal tumors by immunohistochemistry (IHC). Results showed that ZHPV16E7384 affibody had intense and specific staining for E7 oncoprotein in the detected specimen. The E7 oncoprotein was continuously expressed to correspond with the development of precancerous lesions to invasive cervical cancer. IHC staining also revealed that AKT, p-AKT(Ser473), Src and p-Src(Tyr527) proteins were expressed in both patient biopsies and animal tumors, with the highest levels of p-AKT(Ser473)/p-Src(Tyr527) present in invasive cancer. Furthermore, siRNA experiments revealed that HPV16 E7 knockdown significantly impaired expression of p-AKT(Ser473)/p-Src(Tyr527) in both HPV16 E7-positive cancer cells and transformed cells. In addition, transient expression of HPV16 E7 protein promoted significantly expression of p-AKT(Ser473)/p-Src(Tyr527) in primary human keratinocytes. Finally, co-immunoprecipitation analysis proved that HPV 16 E7 protein interacted reciprocally with p-AKT(Ser473)/p-Src(Tyr527). In conclusion, we demonstrate that HPV16 E7 oncoprotein is continuously expressed to promote expression of p-AKT(Ser473)/p-Src(Tyr527) leading to drive the initiation and progression of cervical cancer. Our data provide a novel insight that HPV16 E7 activates p-AKT(Ser473)/p-Src(Tyr527) to establish a mechanistic link between the oncogene and the AKT/Src signaling to trigger cervical carcinogenesis.

Identification of a Schistosoma japonicum MicroRNA That Suppresses Hepatoma Cell Growth and Migration by Targeting Host FZD4 Gene.

Previous studies have demonstrated miRNAs derived from plants and parasites can modulate mammalian gene expression and cell phenotype in a cross-kingdom manner, leading to occurrence of diseases or strengthening resistance of host to diseases such as cancer. In this study, we identified a schistosome miRNA (named Sja-miR-71a) through screening of 57 Schistosoma japonicum miRNAs that exerts antitumor activity in vitro and in vivo models. We demonstrated presence of this parasite miRNA in liver cells during infection. We showed that Sja-miR-71a arrested cell cycle at G0/G1 phase of hepatoma cell lines and inhibited cell proliferation in vitro. The HepG2 transfected with Sja-miR-71a mimics displayed significant reduction of migration and colony formation. Further, growth of the tumor cells transfected with the Sja-miR-71a mimics was obviously suppressed in a xenograft mouse model. Mechanically, we found the antitumor activity of Sja-miR-71a was through targeting a host gene encoding Frizzled Class Receptor 4 (FZD4), as FZD4 small interfering RNAs (siRNAs) generated a similar inhibitory effect on the tumor. These data indicated that Sja-miR-71a is a tumor suppressor miRNA and suggested this parasite-derived miRNA as a potential therapeutic target for cancer.

Dietary mulberry-leaf flavonoids improve the eggshell quality of aged breeder hens.

Eggshell quality is subject to a significant decline in the late laying period, which results in huge economic losses. The purpose of this study was to investigate the effects of dietary mulberry-leaf flavonoids (MF) on the eggshell quality of aged breeder hens. A total of 270 (60-week-old) Qiling breeder hens were randomly assigned to 3 treatments with supplemental dietary MF doses (0, 30, and 60 mg/kg). The results showed that dietary MF improved the eggshell thickness and strength, following the reduced broken egg ratio (P < 0.05). Histological analysis showed that dietary MF increased glandular density and luminal epithelium height in the shell gland (P < 0.05). MF treatment reduced the apoptotic index of the shell gland, following by improved antioxidant capacity (P < 0.05). The protein expression of Caspase 3 was down-regulated, and Nrf2 was up-regulated by dietary MF (P < 0.05). Furthermore, calcium (Ca) content in the serum and shell gland, as well as the activity of Ca2+-ATPase in the shell gland were increased by dietary MF (P < 0.05). Ca transport-related genes (ESRα, ESRß, KCNA1, OPN, CABP-28K and CDH6) in the shell gland were upregulated by dietary MF treatment (P < 0.05). In conclusion, dietary MF could ameliorate the eggshell quality of aged hens by improving antioxidative capability and Ca deposition in the shell gland of uterus.

EBV LMP1-C terminal binding affibody molecule downregulates MEK/ERK/p90RSK pathway and inhibits the proliferation of nasopharyngeal carcinoma cells in mouse tumor xenograft models.

Nasopharyngeal carcinoma (NPC), is an Epstein-Barr virus (EBV) associated malignancy most common in Southern China and Southeast Asia. In southern China, it is one of the major causes of cancer-related death. Despite improvement in radiotherapy and chemotherapy techniques, locoregional recurrence and distant metastasis remains the major causes for failure of treatment in NPC patients. Therefore, finding new specific drug targets for treatment interventions are urgently needed. Here, we report three potential ZLMP1-C affibody molecules (ZLMP1-C15, ZLMP1-C114 and ZLMP1-C277) that showed specific binding interactions for recombinant and native EBV LMP1 as determined by epitope mapping, co-localization and co-immunoprecipitation assays. The ZLMP1-C affibody molecules exhibited high antitumor effects on EBV-positive NPC cell lines and displayed minimal cytotoxicity towards EBV-negative NPC cell line. Moreover, ZLMP1-C277 showed higher antitumor efficacy than ZLMP1-C15 and ZLMP1-C114 affibody molecules. The ability of ZLMP1-C277 decrease the phosphorylation levels of up-stream activator phospho-Raf-1(Ser338), phospho-MEK1/2(Ser217/Ser221), phospho-ERK1/2(Thr202/Thr204), thereby leading to downstream suppression of phospho-p90RSK(Ser380) and transcription factor c-Fos. Importantly, tumor growth was reduced in tumor-bearing mice treated with ZLMP1-C277 and caused no apparent toxicity. Taken together, our findings provide evidence that ZLMP1-C277 as a promising therapeutic agent in EBV-associated NPC.

Novel EBV LMP1 C-terminal domain binding affibody molecules as potential agents for in vivo molecular imaging diagnosis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) latent infection and is common in Southern China and Southeast Asia. The viral latent membrane proteins LMP1 and LMP2 are persistently expressed in NPC tissues; the cytoplasmic domain of LMP1 (LMP1 C-terminal) and LMP2A (LMP2A N-terminal) proteins is essential for maintenance of latency and can alter host cell signaling to facilitate tumor growth and progression. Thus, targeting LMP1 or LMP2 oncoprotein has been an increasing interest for diagnosis and targeted therapy of NPC. Affibody molecules, a new class of small-affinity engineered scaffold proteins, have demonstrated high potential for therapeutics, diagnostics, and biotechnological applications. More recently, radiolabelled HER2-specific affibody molecules have demonstrated to be useful in imaging of HER2 expressing tumor. In this study, we report three novel EBV LMP1 C-terminal (EBV LMP1-C) domain affibody molecules (ZLMP1-C15, ZLMP1-C114, and ZLMP1-C277) were selected by biopanning from a random-peptide displayed phage library and used for molecular imaging in tumor-bearing nude mice. Surface plasmon resonance (SPR), indirect immunofluorescence, and immunohistochemistry (IHC) clearly showed that all three selected affibody molecules have high affinity and specificity in binding to EBV LMP1 protein. Moreover, in vivo tumor imaging revealed that Dylight-755-labeled affibody molecules accumulated rapidly in tumor site after injection (1 h) and then were continuously maintained for 24 h in EBV-positive NPC xenograft mice model. In conclusion, our findings highlight the potential use of ZLMP1-C affibody molecules as tumor-specific molecular imaging agents of EBV-associated NPC.Key points• We screened three novel affibody molecules (ZLMP1-C15, ZLMP1-C114, and ZLMP1-C277) targeting EBV LMP1-C terminal domain• ZLMP1-C recognize the recombinant and native LMP1-C with high affinity and specificity• ZLMP1-C can be used for molecular imaging.

A Schistosoma japonicum MicroRNA Exerts Antitumor Effects Through Inhibition of Both Cell Migration and Angiogenesis by Targeting PGAM1.

MicroRNA (miRNA) is an important regulator for gene expression. Recent studies showed that some heterogenous miRNAs derived from both parasite and plant can regulate expression of mammalian gene in a cross-species or even a cross-kingdom manner. Here, we identified a Schistosoma japonicum miRNA (designated as sja-miR-61) that is present in the hepatocyte of mice infected with the parasite. The sja-miR-61 mimics significantly inhibited the migration of both mouse and human hepatoma cells in vitro. In a xenograft animal model, significant reductions of the tumor volume and weight were observed in mice inoculated with hepatoma cells transfected with sja-miR-61 mimics compared to the controls. We found that the in vivo inhibition of tumor growth was through its anti-angiogenesis activity. Mechanically, we identified the phosphoglycerate mutase 1 (PGAM1) gene as a target of sja-miR-61 and found that the sja-miR-61-mediated suppression of cell migration and anti-angiogenesis by cross-species down-regulation of PGAM1 expression. These data indicated that sja-miR-61 is a tumor suppressor miRNA that may have therapeutic potential for human cancers.

Novel Affibody Molecules Targeting the HPV16 E6 Oncoprotein Inhibited the Proliferation of Cervical Cancer Cells.

Despite prophylactic vaccination campaigns, high-risk human papillomavirus (HPV)-induced cervical cancer remains a significant health threat among women, especially in developing countries. The initial occurrence and consequent progression of this cancer type primarily rely on, E6 and E7, two key viral oncogenes expressed constitutively, inducing carcinogenesis. Thus, E6/E7 have been proposed as ideal targets for HPV-related cancer diagnosis and treatment. In this study, three novel HPV16 E6-binding affibody molecules (ZHPV16E61115, ZHPV16E61171, and ZHPV16E61235) were isolated from a randomized phage display library and cloned for bacterial production. These affibody molecules showed high binding affinity and specificity for recombinant and native HPV16 E6 as determined by surface plasmon resonance, indirect immunofluorescence, immunohistochemistry, and near-infrared small animal optical imaging in vitro and in vivo. Moreover, by binding to HPV16 E6 protein, ZHPV16E61235 blocked E6-mediated p53 degradation, which increased the expression of some key p53 target genes, including BAX, PUMA and p21, and thereby selectively reduced the viability and proliferation of HPV16-positive cells. Importantly, ZHPV16E61235 was applied in combination with HPV16 E7-binding affibody ZHPV16E7384 to simultaneously target the HPV16 E6/E7 oncoproteins, and this combination inhibited cell proliferation more potently than either modality alone. Mechanistic studies revealed that the synergistic antiproliferative activity depends primarily on the induction of cell apoptosis and senescence but not cell cycle arrest. Our findings provide strong evidence that three novel HPV16 E6-binding affibody molecules could form a novel basis for the development of rational strategies for molecular imaging and targeted therapy in HPV16-positive preneoplastic and neoplastic lesions.

Replacing fish oil and astaxanthin by microalgal sources produced different metabolic responses in juvenile rainbow trout fed 2 types of practical diets.

Dietary fish oil supplementation provides n-3 long-chained polyunsaturated fatty acids for supporting fish growth and metabolism and enriching fillet with eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; c22:6n-3). Two experiments were performed as a 3 × 2 factorial arrangement of dietary treatments for 16 wk to determine effects and mechanisms of replacing 0%, 50%, and 100% fish oil with DHA-rich microalgae in combination with synthetic vs. microalgal source of astaxanthin in plant protein meal (PM)- or fishmeal (FM)- based diets for juvenile rainbow trout (Oncorhynchus mykiss). Fish (22 ± 0.26 g) were stocked at 17/tank and 3 tanks/diet. The 100% fish oil replacement impaired (P < 0.0001) growth performance, dietary protein and energy utilization, body indices, and tissue accumulation of DHA and EPA in both diet series. The impairments were associated (P < 0.05) with upregulation of hepatic gene expression related to growth (ghr1and igf1) and biosynthesis of DHA and EPA (fads6 and evol5) that was more dramatic in the FM than PM diet-fed fish, and more pronounced on tissue EPA than DHA concentrations. The source of astaxanthin exerted interaction effects with the fish oil replacement on several measures including muscle total cholesterol concentrations. In conclusion, replacing fish oil by the DHA-rich microalgae produced more negative metabolic responses than the substitution of synthetic astaxanthin by the microalgal source in juvenile rainbow trout fed 2 types of practical diets.

Correction: Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

[Expression of HPV16 E6 recombinant protein and preparation of its rabbit polyclonal antibody].

Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.

Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) induced by latent infection with Epstein-Barr virus (EBV) remains the most common head and neck cancer in Southeast Asia, especially in the southern part of China. It is well known that persistent expression of two EBV latent membrane proteins (LMP1/LMP2A) plays a key role in nasopharyngeal carcinogenesis. Therefore, the therapeutic approach of targeting the LMP1/LMP2A protein and subsequently blocking the LMP1/LMP2A-mediated signalling pathway has been considered for treating patients with NPC. Recently, affibody molecules, a new class of small (~6.5 kDa) affinity proteins, have been confirmed to be powerful generalisable tools for developing imaging or therapeutic agents by targeting specific molecules. In this study, three EBV LMP2A N-terminal domain-binding affibody molecules (ZLMP2A-N85, ZLMP2A-N110 and ZLMP2A-N252) were identified by screening a phage-displayed peptide library, and their high affinity and specificity for the EBV LMP2A N-terminal domain were confirmed by surface plasmon resonance (SPR), indirect immunofluorescence, co-immunoprecipitation and near-infrared small animal fluorescence imaging in vitro and in vivo. Moreover, affibody molecules targeting the EBV LMP2A N-terminal domain significantly reduced the viability of the EBV-positive cell lines C666-1, CNE-2Z and B95-8. Further investigations showed that affibody ZLMP2A-N110 could inhibit the phosphorylation of AKT, GSK-3ß and ß-catenin signalling proteins, leading to suppression of ß-catenin nuclear translocation and subsequent inhibition of c-Myc oncogene expression, which may be responsible for the reduced viability of NPC-derived cell lines. In conclusion, our findings provide a strong evidence that three novel EBV LMP2A N-terminal domain-binding affibody molecules have great potential for utilisation and development as agents for both molecular imaging and targeted therapy of EBV-related NPC.

Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.

Cross-Species Suppression of Hepatoma Cell Growth and Migration by a Schistosoma japonicum MicroRNA.

Schistosoma japonicum eggs trapped in host liver secretes microRNA (miRNA)-containing extracellular vesicles (EVs) that can be transferred to host cells. Recent studies demonstrated that miRNAs derived from plants can modulate gene expression and phenotype of mammalian cells in a cross-kingdom manner. In this study, we identified a Schistosoma japonicum miRNA (e.g., Sja-miR-3096) that is present in the hepatocytes of mice infected with the parasite and has notable antitumor effects in both in vitro and in vivo models. The Sja-miR-3096 mimics suppressed cell proliferation and migration of both murine and human hepatoma cell lines by targeting phosphoinositide 3-kinase class II alpha (PIK3C2A). We generated a murine hepatoma cell line that stably expressed the pri-Sja-miR-3096 gene and demonstrated cross-species processing of the schistosome pri-miRNA to the mature Sja-miR-3096 in the mammalian cell. Importantly, inoculation of this cell line into the scapula and livers of mice led to a complete suppression of tumorigenesis of the hepatoma cells. Moreover, tumor weight was significantly reduced on intravenous administration of Sja-miR-3096 mimics. Thus, the schistosome miRNA-mediated antitumor activity occurs in host liver cells during schistosome infection, which may strengthen resistance of host to liver cancer, and discovery and development of such miRNAs may present promising interventions for cancer therapy.

Schistosoma japonicum MiRNA-7-5p Inhibits the Growth and Migration of Hepatoma Cells via Cross-Species Regulation of S-Phase Kinase-Associated Protein 2.

MicroRNAs (miRNAs) play important roles in human diseases, such as cancer. Human miRNA-7-5p is a tumor suppressor miRNA that inhibits tumor growth by regulating multiple oncogenic signal pathways. Recently, studies revealed that plant miRNAs could regulate mammalian gene expression in a cross-kingdom manner. Schistosoma japonicum miRNA-7-5p (designated as sja-miR-7-5p) is conserved between the parasites and mammals. Thus, we investigated whether sja-miR-7-5p has similar antitumor activity to its mammalian counterpart. We first showed that sja-miR-7-5p was detected in host hepatocytes during S. japonicum infection. The sja-miR-7-5p mimics significantly inhibited the growth, migration, and colony formation of mouse and human hepatoma cell lines in vitro, and induced G1/G0 cell cycle arrest. In a xenograft animal model, the tumor volume and weight were significantly reduced in mice inoculated with hepatoma cells transfected with sja-miR-7-5p mimics compared with those transfected with NC miRNAs. Furthermore, the antitumor activity of sja-miR-7-5p was suggested by cross-species downregulation of the S-phase kinase-associated protein 2 gene in the host. Thus, sja-miR-7-5p is translocated into hepatocytes and exerts its anti-cancer activities in mammals, implying that sja-miR-7-5p might strengthen host resistance to hepatocellular carcinoma during schistosome infection.

A high-risk papillomavirus 18 E7 affibody-enabled in vivo imaging and targeted therapy of cervical cancer.

High-risk papillomavirus (HPV) is one of the major reasons for cervical cancer, causing most lethal gynecologic malignancies worldwide. For cervical cancer progression, oncogene E7 plays vital roles and is used as one of the major targets for cervical tumor diagnosis and treatment. In the clinic, successful treatment of cervical cancer relies on diagnosing the disease at an early stage, where a late-stage diagnosis usually led to treatment failure. In this work, we designed and purified an HPV18 E7 oncogene targeting affibody, named as ZHPV18E7, for in vitro and in vivo imaging and targeted treatment of cervical cancer. In vitro, ZHPV18E7 showed a specific targeting effect against an HPV18 positive cell line; as a contrast, the affibody did not target the HPV18 negative cell line. In vivo, we tested the bio-distribution of the affibody in mice bearing cervical cancer. The whole animal imaging analysis indicated the affibody-targeted tumor tissue specifically with 10 min after injection, and the affibody reached the highest level at tumor tissues 45 min after injection. At the 24th hour after injection, the affibody still maintained a certain level in tumor tissues compared to other organs. To test the therapeutic effect of this affibody, we modified the affibody (i.e., ZHPV18E7) with a clinically used anti-cancer agent (i.e., Pseudomonas exotoxin). In a mice cervical cancer model, ZHPV18E7 was able to deliver Pseudomonas exotoxin to tumor tissues effectively, showing great potential for cancer treatment. This study indicated that ZHPV18E7 could be employed for in vitro imaging and targeted treatment of cervical cancer. Beyond the chemotherapeutic agent used in this work, the affibody could be extended for carrying other therapeutic agents for cervical cancer treatment.

A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer.

Cervical cancer, the second most common cause of cancer death in women worldwide, is significantly associated with infection of high-risk human papillomaviruses (HPVs), especially the most common genotype, HPV 16. To date, there is no established noninvasive therapy to treat cervical cancer. Methods: Here, we report a novel affitoxin that targets HPV16 E7 protein, one of the primary target proteins in molecular targeted therapy for HPV-induced cervical cancer. The affitoxin, ZHPV16E7 affitoxin384 was generated by fusing the modified Pseudomonas Exotoxin A (PE38KDEL) to the HPV16 E7-specific affibody. The expressed and purified ZHPV16E7 affitoxin384 was characterized using numerous methods. SPR assay, indirect immunofluorescence assay, and near-infrared (NIR) optical imaging were respectively performed to assess the targeting ability of ZHPV16E7 affitoxin384 to HPV16 E7 protein both in vitro and in vivo. Cell viability assays and SiHa tumor-bearing nude mice were used to evaluate the efficacy of ZHPV16 E7 affitoxin384 in vitro and in vivo, respectively. Results: Using in vitro methods the SPR assay and indirect immunofluorescence assay showed that ZHPV16E7 affitoxin384 targeted HPV16 E7 with high binding affinity and specificity. Significant reduction of cell viability in HPV16 positive cells was observed in the presence of ZHPV16 E7 affitoxin384. By NIR optical imaging, ZHPV16 E7 affitoxin384 specifically targeted HPV16 positive tumors in vivo. ZHPV16E7 affitoxin384 showed significant in vivo antitumor efficacy in two kinds of tumor-bearing nude mouse models. Conclusions: ZHPV16E7 affitoxin384 is a potent anti-cervical cancer therapeutic agent that could be effective against HPV16 positive tumors in humans.

Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer.

High-risk human papillomavirus (HPV16 and HPV18) are now widely recognized as responsible for cervical cancer, which remains to be the most common gynecologic malignancy in women worldwide. It is well known that viral oncoproteins E6/E7 play key roles in HPV-associated cervical carcinogenesis. Thus, in vivo detection of the two oncoproteins may provide important diagnostic information influencing patient management. More recently, affibody molecules have been demonstrated to be a promising candidate for development as molecular imaging probes. Based on the two monomeric affibody molecules (ZHPV16E7 and ZHPV18E7) generated in our laboratory, here, we used a peptide linker (Gly4Ser)3 to link ZHPV16E7 and ZHPV18E7 to develop a novel heterodimeric affibody ZHPV16E7-(Gly4Ser)3-ZHPV18E7. Both biosensor and immunofluorescence assays have proved that the heterodimeric affibody molecule targeted simultaneously HPV16 and HPV18E7 proteins by binding to the viral oncoproteins. In vivo tumor-imaging experiments using the Dylight755-labeled heterodimeric affibody revealed that strongly high-contrast tumor retention of the heterodimers occurred in both HPV16- and HPV18-derived tumors of nude mice 0.5 h post-injection. The accumulation of Dylight755-labeled heterodimers in tumors was achieved over 48 h. Therefore, we believe that this novel heterodimeric affibody molecule has great potential utility in molecular imaging in vivo and diagnosis of HPV-associated cervical cancers.