A peptide-salinomycin conjugate with a bystander effect reduces the stemness characteristics of ovarian cancer cells and enhances drug sensitivity.

Salinomycin (Sal) has attracted considerable attention in the field of tumor treatment, especially for its inhibitory effect on cancer stem cells (CSCs) and drug-resistant tumor cells. However, its solubility and targeting specificity pose significant challenges to its pharmaceutical development. Sal-A6, a novel peptide-drug conjugate (PDC), was formed by linking the peptide A6 targeting the CSC marker CD44 with Sal using a specific linker. This conjugation markedly enhances the physicochemical properties of Sal and compared to Sal, Sal-A6 demonstrated a significantly increased activity against ovarian cancer. Furthermore, Sal-A6, employing a disulfide bond as a linker, exhibited bystander killing effect. Moreover, it induces substantial cytotoxic effect on both cancer stem cells and drug-resistant cells in addition to enhance chemosensitivity of resistant ovarian cancer cells. In summary, the results indicated that Sal-A6, a novel PDC derived from Sal, has potential therapeutic applications in the treatment of ovarian cancer and drug-resistant patients. Additionally, this discovery offers insights for developing PDC-type drugs using Sal as a foundation.

A multifunctional antibody fusion protein 57103 targeting CD24, IL-4R, and αvß3 for treating cancer and regulating the tumor microenvironment.

Cancer is one of the top 10 fatal diseases worldwide, among which advanced metastatic carcinoma has the highest mortality rate. Sunitinib and immune checkpoint blockers are commonly used to treat metastatic renal carcinoma with limited efficacy. Therefore, there is an urgent need to develop novel targeted therapies for metastatic renal cancer. In this study, we designed an antibody fusion protein, 57103, that simultaneously targeted the cluster of differentiation 24 (CD24), interleukin 4 receptor (IL-4R), and integrin receptors αvß3 and α5ß1. In vitro assays showed that 57103 significantly suppressed the proliferation, migration, invasion, colony formation, and adhesion abilities of renal cancer cells, resulting in a comprehensive and significant antitumor effect. Furthermore, 57103 inhibited angiogenesis, promoted THP1-derived M0-type macrophage phagocytosis, and enhanced the antibody-dependent cellular cytotoxicity of peripheral blood mononuclear and NK92MI-CD16a cells. In vivo experiments revealed significant inhibition of tumor growth in ACHN cell xenograft nude mice and an MC38-hCD24 tumor-bearing mouse model. Immunohistochemical analysis showed that 57103 decreased the proliferation and induced the apoptosis of renal cancer cells, while inhibiting angiogenesis. The MC38-hPDL1 and MC38-hCD24-hPDL1 tumor-bearing mouse models further offer the possibility of combining 57103 with the PDL1 antagonist atezolizumab. In conclusion, 57103 is a potential candidate drug for the treatment of metastatic renal carcinoma or PDL1-overexpressing cancer.

The peptide Acein promotes dopamine secretion through clec-126 to extend the lifespan of elderly C. elegans.

Dopamine plays a crucial role in regulating brain activity and movement and modulating human behavior, cognition and mood. Regulating dopamine signaling may improve cognitive abilities and physical functions during aging. Acein, a nonapeptide of sequence H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH is able to stimulate dopamine secretion in the brain. By using genetic editing and lifespan investigation in C. elegans, we showed that the lack of the C-type lectin domain-containing protein clec-126 significantly suppressed the aging phenotype and prolonged lifespan, while overexpression of clec-126 promoted aging-related phenotypes and accelerated the aging process. We examined the aging phenotype of C. elegans and showed that Acein could induce a decrease in clec-126 expression, prolonging the lifespan of aged C. elegans. The mechanism proceeds through the Acein-induced stimulation of dopamine secretion that ameliorates motor function decline and extends the healthy lifespan of aged C. elegans. In addition, we also observed an increase in brood number. Our study has shown that Acein regulates dopamine secretion and has good antiaging activity by decreasing clec-126 expression.

Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.

BACKGROUND: Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma. METHODS: The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways. RESULTS: The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects. CONCLUSIONS: This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.

Trends in insulin resistance: insights into mechanisms and therapeutic strategy.

The centenary of insulin discovery represents an important opportunity to transform diabetes from a fatal diagnosis into a medically manageable chronic condition. Insulin is a key peptide hormone and mediates the systemic glucose metabolism in different tissues. Insulin resistance (IR) is a disordered biological response for insulin stimulation through the disruption of different molecular pathways in target tissues. Acquired conditions and genetic factors have been implicated in IR. Recent genetic and biochemical studies suggest that the dysregulated metabolic mediators released by adipose tissue including adipokines, cytokines, chemokines, excess lipids and toxic lipid metabolites promote IR in other tissues. IR is associated with several groups of abnormal syndromes that include obesity, diabetes, metabolic dysfunction-associated fatty liver disease (MAFLD), cardiovascular disease, polycystic ovary syndrome (PCOS), and other abnormalities. Although no medication is specifically approved to treat IR, we summarized the lifestyle changes and pharmacological medications that have been used as efficient intervention to improve insulin sensitivity. Ultimately, the systematic discussion of complex mechanism will help to identify potential new targets and treat the closely associated metabolic syndrome of IR.

Cancer-related micropeptides encoded by ncRNAs: Promising drug targets and prognostic biomarkers.

An exciting emerging topic in the noncoding RNA (ncRNA) field is the discovery of short peptides called micropeptides (≤100 amino acids), whose novel therapeutic opportunities remain under-explored. Micropeptides have been suggested to play essential regulatory roles in diverse species of physiological and pathological processes. Genomics studies have revealed that these micropeptides are encoded by small open reading frames (sORFs) concealed in misannotated ncRNAs, generally lncRNAs (long noncoding RNAs) and circRNAs (circular RNAs). These ncRNA-encoded micropeptides have been shown to contribute to tumorigenesis but little is known about their pathological mechanism because of challenges in translated sORF identification techniques. Here, we review the best-validated micropeptides involved in the progression of human tumors and discuss their therapeutic and/or prognostic potential, at the same time, we also give our own suggestions on the concept of potential-coding RNA and micropeptides.

Integrins as attractive targets for cancer therapeutics.

Integrins are transmembrane receptors that have been implicated in the biology of various human physiological and pathological processes. These molecules facilitate cell-extracellular matrix and cell-cell interactions, and they have been implicated in fibrosis, inflammation, thrombosis, and tumor metastasis. The role of integrins in tumor progression makes them promising targets for cancer treatment, and certain integrin antagonists, such as antibodies and synthetic peptides, have been effectively utilized in the clinic for cancer therapy. Here, we discuss the evidence and knowledge on the contribution of integrins to cancer biology. Furthermore, we summarize the clinical attempts targeting this family in anti-cancer therapy development.

[Corrigendum] In vitro and in vivo activities of an antitumor peptide HM-3: A special dose-efficacy relationship on an HCT­116 xenograft model in nude mice.

After the publication of the article, the authors have realized that Figs. 3 and 7 in their paper were published with errors; in the first instance, regarding Fig. 3, panels 'C' and 'D' contained partially overlapping data and were derived from the same original source, where these images were intended to show the effect of 2 ng/ml sunitinib and 2 µg/ml HM­3, respectively, on cell migration. Likewise, in Fig. 7, panels 'C' and 'D' also contained partially overlapping data derived from the same original source, even though these images were intended to show representative images for sections of tumor tissue from the HM­3 (3 mg/kg) and HM­3 (48 mg/kg) treatment groups. These errors arose inadvertently, as a consequence of the authors' mishandling of their data. The revised versions of Figs. 3 and 7, featuring the corrected data panels for panels 'C' and 'D' in both Figures, are shown opposite. The revised data shown for these Figures do not affect the overall conclusions reported in the paper. The authors apologize to the Editor of Oncology Reports and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 36: 2951­2959, 2016; DOI: 10.3892/or.2016.5077].

Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide.

BACKGROUND: Chimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. However, excessive activity and poor control over such engineered T cells cause significant safety challenges, such as cytokine release syndrome and organ toxicities. To enhance the specificity and controllable activity of CAR-T cells, we report a novel switchable dual-receptor CAR-engineered T (sdCAR-T) cell and a new switch molecule of FITC-HM-3 bifunctional molecule (FHBM) in this study. METHODS: We designed a fusion molecule comprising FITC and HM-3. HM-3, an antitumor peptide including an Arg-Gly-Asp sequence, can specifically target integrin αvß3 that is presented on some tumor cells. Moreover, to improve the specificity of CAR-T cells, we also generated the sdCAR-T cell line against cognate tumor cells expressing human mesothelin (MSLN) and integrin αvß3. Finally, the activity of sdCAR-T cell and FHBM is verified via in vitro and in vivo experiments. RESULTS: In the presence of FHBM, the designed sdCAR-T cells exerted high activity including activation and proliferation and had specific cytotoxicity in a time- and dose-dependent manner in vitro. Furthermore, using a combination of FHBM in nude mice, sdCAR-T cells significantly inhibited the growth of MSLN+ K562 cells and released lower levels of the cytokines (e.g., interleukin-2, interferon γ, interleukin-6, and tumor necrosis factor α) relative to conventional CAR-T cells, obtaining specific, controllable, and enhanced cytotoxicity. CONCLUSIONS: Our data indicate that FHBM can accurately control timing and dose of injected CAR-T cells, and sdCAR-T cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvß3. Therefore, combination therapies using sdCAR-T cells and the switch molecule FHBM have significant potential to treat malignancies.

Novel production method of innovative antiangiogenic and antitumor small peptides in Escherichia coli.

BACKGROUND: Developing innovative drugs with potent efficacy, specificity, and high safety remains an ongoing task in antitumor therapy development. In the last few years, peptide drugs have become attractive agents in cancer therapy. HM-3, mainly with antiangiogenic effect, and AP25, with an additional antiproliferative effect, are two peptides designed in our laboratory targeting αvß3 and α5ß1 integrins, respectively. The low molecular weight of the two peptides renders their recombinant expression very difficult, and the complicated structure of AP25 makes its chemical synthesis restricted, which presents a big challenge for its development. METHODS: Bifunctional peptides designed by the ligation of HM-3 and AP25, using linkers with different flexibility, were prepared using recombinant DNA technology in Escherichia coli. The fusion peptides were expressed in a modified auto-induction medium based on a mixture of glucose, glycerol, and lactose as carbon substrates and NH4+ as nitrogen source without any amino acid or other elements. Subsequently, the antiangiogenic, antiproliferative, and cell adhesion assays were conducted to evaluate the bioactivity of the two fusion peptides. RESULTS: The peptides were successfully expressed in a soluble form without any induction, which allows the culture to reach higher cell density before protein expression occurs. Human umbilical vein endothelial cell migration assay and chick embryo chorioallantoic membrane assay showed, at low doses, a significantly increased antiangiogenic effect (>75%) of the purified products compared with the single molecules. Meanwhile, MTT assay confirmed their enhanced antitumor activity against gastric cancer cell line MGC-803; however, no significant effect was observed on hepatoma HepG2 cells and no cytotoxicity on normal human lens epithelial cell SRA01/04 and human epithelial esophageal cells. CONCLUSION: Bifunctional molecules with antiangiogenic and antiproliferative effects were obtained by using this technique, which presents an alternative for small peptide production, instead of the conventional chemical method. The increased molecular weight facilitates the peptide expression with a simultaneous improvement in their stability and biological activity.

Tumor-related interleukins: old validated targets for new anti-cancer drug development.

In-depth knowledge of cancer molecular and cellular mechanisms have revealed a strong regulation of cancer development and progression by the inflammation which orchestrates the tumor microenvironment. Immune cells, residents or recruited, in the inflammation milieu can have rather contrasting effects during cancer development. Accumulated clinical and experimental data support the notion that acute inflammation could exert an immunoprotective effect leading to tumor eradication. However, chronic immune response promotes tumor growth and invasion. These reactions are mediated by soluble mediators or cytokines produced by either host immune cells or tumor cells themselves. Herein, we provide an overview of the current understanding of the role of the best-validated cytokines involved in tumor progression, IL-1, IL-4 and IL-6; in addition to IL-2 cytokines family, which is known to promote tumor eradication by immune cells. Furthermore, we summarize the clinical attempts to block or bolster the effect of these tumor-related interleukins in anti-cancer therapy development.

Combination Therapy of PEG-HM-3 and Methotrexate Retards Adjuvant-Induced Arthritis.

At present, the early phenomenon of inflammatory angiogenesis is rarely studied in Rheumatoid arthritis (RA). Previous research found that PEG-HM-3, an integrin inhibitor, possessed anti-angiogenesis and anti-rheumatic activity. In this study, the advantages of inhibiting angiogenesis and immune cell adhesion and migration, as well as the benefits of anti-arthritis effects, were evaluated using a combination of PEG-HM-3 and methotrexate (MTX). In vitro, spleen cell proliferation and the levels of tumor necrosis factor α (TNF-α) in macrophage supernatant were assessed. Hind paw edema, arthritis index, clinical score, body weight and immunohistochemistry (IHC) of the spleen, thymus, and joint cavity were evaluated in vivo in adjuvant-induced arthritis rats. Joints of the left hind paws were imaged by X-ray. The expression of the toll-like receptor 4 (TLR-4) protein was assessed in lipopolysaccharide (LPS)-induced synoviocytes. PEG-HM-3 combined with MTX significantly reduced primary and secondary swelling of the hind paws, the arthritis index, the clinical score and bone erosion. The results of IHC showed that the levels of interleukin-6 (IL-6) in spleens and the levels of TNF-α, CD31 (cluster of differentiation 31), and CD105 in the joint cavity were decreased. The body weight of rats was maintained during combination therapy. Ankle cavity integrity, and bone erosion and deformity were improved in combination treatment. The expression of TLR-4 was significantly reduced with combination treatment in rat synoviocytes. Co-suppression of both inflammation and angiogenesis in arthritis was achieved in this design with combination therapy. The activity of nuclear transcription factor (NF-κB) and the expression of inflammatory factors were down regulated via integrin αvß3 and TLR-4 signaling pathways. In the future, the application of this combination can be a candidate in early and mid-term RA therapy.

In vitro and in vivo activities of an antitumor peptide HM-3: A special dose-efficacy relationship on an HCT­116 xenograft model in nude mice.

Anti-angiogenesis is an important therapy for cancer treatment. Peptide HM-3 is an integrin antagonist with anti-angiogenic and antitumor activity. Previous research found that HM-3 at an effective dose inhibited tumor growth whereas at higher doses, the inhibitory effect gradually decreased. In the present study, three human tumor cell lines, human colorectal cancer cell (HCT-116) and human hepatic cancer cell (Hep G-2 and SMMC-7721), were selected and their interactions with HM-3 were compared with western blot and flow cytometric assays. The effect of HM-3 on the migration of two tumor cell lines (HCT-116 and Hep G-2) was also evaluated and a bell-shaped dose-efficacy curve was found for both cell lines. Furthermore, in vivo imaging in BALB/c nude mice confirmed that HM-3 had a short half-life and targeted the tumor tissue. Moreover, on an HCT-116 xenograft model in BALB/c nude mice, HM-3 at 3 mg/kg inhibited tumor growth with an inhibition rate of 71.5% (by tumor mass) whereas at 12 and 48 mg/kg, the inhibition rates were 59.2 and 36.0%, respectively. Immunohistochemistry analyses found that both sunitinib (60 mg/kg) and HM-3 (3 and 48 mg/kg) decreased microvascular density and increased percent of HIF-1α and VEGF expressing cells. The present study investigated the effect of tumor microenvironments on the antitumor effect of HM-3 and concluded that HM-3 inhibited angiogenesis and thereafter tumor growth by directly inhibiting HUVEC migration. The special dose-efficacy curves for antitumor effect and for cell migration inhibition were correlated. The present study also confirmed that the effective dose has to be strictly defined for better clinical applications of anti­angiogenic drugs such as HM-3.

Enrichment and characterization of cancer stem cells from a human non-small cell lung cancer cell line.

Tumor cells from the same origin comprise different cell populations. Among them, cancer stem cells (CSCs) have higher tumorigenicity. It is necessary to enrich CSCs to determine an effective way to suppress and eliminate them. In the present study, using the non-adhesive culture system, tumor spheres were successfully generated from human A549 non-small cell lung cancer (NSCLC) cell line within 2 weeks. Compared to A549 adherent cells, sphere cells had a higher self-renewal ability and increased resistance to cytotoxic drugs. Sphere cells were more invasive and expressed stem cell markers including octamer­binding transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) at high levels. CD133, a disputed marker of lung CSCs, was also upregulated. Tumor sphere cells showed higher tumorigenic ability in vivo, indicating that more CSCs were enriched in the sphere cells. More blood vessels were formed in the tumor generated by sphere cells suggesting the interaction between CSCs and blood vessel. A reliable model of enriching CSCs from the human A549 NSCLC cell line was established that was simple and cost-effective compared to other methods.

Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli.

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.