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Epidermal growth factor receptor (EGFR) is an efficient target for cancer therapy. In this study, a high-affinity EGFR-antagonistic affibody (ZEGFR) molecule coupled with cisplatin-loaded PEGylated liposomes (LS-DDP) was applied to actively target EGFR+ A431 tumor cells in vitro and in vivo. The LS-DDP coupled with ZEGFR (AS-DDP) had an average size of 140.01 ± 0.84 nm, low polydispersity, a zeta potential of -13.40 ± 0.8 mV, an acceptable encapsulation efficiency of 17.30 ± 1.35%, and released cisplatin in a slow-controlled manner. In vitro, AS-DDP demonstrated a higher amount of platinum intracellular uptake by A431 cells than LS-DDP. The IC50 value of AS-DDP (9.02 ± 1.55 μg/ml) was much lower than that of LS-DDP (16.44 ± 0.87 μg/ml), indicating that the anti-tumor effects of AS-DDP were remarkable due to the modification of ZEGFR. In vivo, the concentration of AS-DDP in the tumor site increased more than 1.76-fold, while an increase in apoptotic cells at 48 h compared to the LS-DDP was also observed, illustrating that AS-DDP possessed excellent tumor-targeting efficiency. As a result, the targeted nano-liposomes achieved greater tumor suppression. Therefore, selective targeting of LS-DDP coupled with ZEGFR enhanced the anti-tumor effects and appeared to be a promising strategy for the treatment of EGFR+ tumors.

A series of glutathione (GSH)-responsive polydopamine (PDA) nanoparticles (NPs) were prepared using a disulfide-linked dopamine dimer as starting material, of which the size could be tuned systematically by adjusting the amount of ammonia solution used. Molecules of a phthalocyanine (Pc)-based photosensitiser and an epidermal growth factor receptor (EGFR)-targeting peptide were then sequentially immobilised on the surface of the NPs through coupling with the surface functionalities of PDA. The immobilised Pc molecules in the resulting nanosystem were photodynamically inactive due to the severe self-quenching effect and the quenching by the PDA core. Upon exposure to GSH in phosphate-buffered saline or EGFR-positive cancer cells, namely A549 and A431 cells, the NPs were disassembled through cleavage of the disulfide linkages to release the Pc molecules, thereby restoring their fluorescence emission and singlet oxygen generation. The NPs with the smallest size (ca. 200 nm in diameter) exhibited the highest cellular uptake and high photocytotoxicity with IC50 values as low as 0.05 μM based on Pc. These NPs could also accumulate and be activated in the tumour of A431 tumour-bearing nude mice, lighting up the tumour with fluorescence over a period of 72 h and completely eradicating the tumour through laser irradiation for 10 min (675 nm, 20 J cm-2). The results suggest that these biodegradable and versatile PDA-based NPs can serve as a promising nanoplatform for fabrication of advanced photosensitisers for targeted photodynamic therapy.

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2′-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer – EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

The therapeutic effect of dihydroartemisinin (DHA) against cutaneous squamous cell carcinoma (cSCC) has been previously demonstrated; however, the underlying mechanism remains unclear. This study sought to verify the therapeutic effect of DHA against cSCC and explore its underlying mechanism in A431 cSCC cells. This study reported that DHA inhibited A431 cells proliferation in a time- and concentration-dependent manner and promoted A431 cells apoptosis. Moreover, DHA inhibited the invasion and migration of A431 cells. Mechanistically, DHA promoted autophagy and inhibited activation of the absent in melanoma 2 (AIM2) inflammasome pathway and NF-κB/HIF-1α/VEGF pathway. Treatment of A431 cells with the mTOR inhibitor, and autophagy promoter, rapamycin also inhibited these two pathways. In conclusion, DHA inhibited activation of the AIM2 inflammasome pathway and NF-κB/HIF-1α/VEGF pathway by promoting autophagy in A431 cells, thus accounting for its therapeutic effect. Induction of autophagy by DHA may be mediated by inhibiting the mTOR pathway and promoting reactive oxygen species production.

Geraniol, an acyclic monoterpene present in several plant species' essential oils, is utilized as a food additive. It possesses potent antiproliferative and antitumor effects ascribed to its antiinflammatory, and antioxidant properties. The study aimed to understand geraniol's mechanism in human lung and skin cancer cells by employing molecular and cell target-based assays. SRB, NRU, MTT assays, qRT-PCR, molecular docking, and EAC model were used. Geraniol inhibits the proliferation of PC-3, A431, and A549 cells (~50%) and suppresses the activity of ornithine decarboxylase (15.42 ± 0.61 μM) and hyaluronidase (57.61 ± 8.53 μM) in A549 cells; LOX-5 (25.44 ± 3.50 μM) and hyaluronidase (90.71 ± 2.38 μM) in A431 cells. The qRT-expression analysis of the targeted gene depicts non-significant change at the transcriptional level of LOX-5 in A431 cells. A robust binding interaction of geraniol with molecular targets was observed in the molecular docking studies. In Ehrlich Ascites Carcinoma model, geraniol inhibit tumor growth by 50.08% at 75 mg/kg bw and was found to be safe up to 1,000 mg/kg bw in a toxicity study. Geraniol has two prenyl units allied head-to-tail and functionalized with one hydroxyl group at its tail end could be responsible for the antiproliferative activity. These observations provide evidence for geraniol to be used as a new prototype to develop a novel anticancer agent.

The research on the morphological and mechanical properties of single cells has provided a crucial way of understanding the cellular physiology and metabolism. In this study, the selective anticancer effects of Phellinus linteus on A431 and HaCat cells and their morphological and mechanical properties were systematically investigated by atomic force microscopy (AFM). Notably, the cell morphology on the micronano scale was observed under both the physiological environment and immobilization conditions. The significant morphological changes of A431 cells from the flat to spherical shape, the increase of cell height, and the decrease of the particles on the cell membrane were confirmed to be related to the cell apoptosis under the treatment of the Phellinus linteus water extract (PLWE). Moreover, the small morphology variations of HaCat cells showed that the PLWE presented a high anticancer effect on A431 cells but low toxicity on HaCat cells, which indicated a potential cell selectivity between cancer and normal cells. This work proved that Phellinus linteus could be used as a potential candidate for selective anticancer treatments.

Sphingosine kinase 1 (SphK1) is overexpressed in skin squamous cell carcinoma (SCC). It has emerged as a novel therapeutic oncotarget. The current study identified a novel SphK1-targeting microRNA, microRNA-6784 (miR-6784). Here, we show that miR-6784 is located at the cytoplasm of A431 skin SCC cells. It directly binds to SphK1 mRNA. Ectopic overexpression of miR-6784 inhibited SphK1 3’-untranslated region (UTR) luciferase activity and downregulated its expression. Moreover, miR-6784 overexpression caused ceramide accumulation in skin SCC cells. Functional studies in established (A431 and SCC9) and primary skin SCC cells revealed that miR-6784 overexpression inhibited cell viability, proliferation, migration, and invasion. It also simultaneously provoked apoptosis activation. Conversely, miR-6784 silencing by antagomiR-6784 induced SphK1 elevation and augmented A431 cell proliferation, migration, and invasion. miR-6784 overexpression-induced anti-A431 cell activity was inhibited by the expression of an UTR-null SphK1 construct. CRISPR/Cas9-induced SphK1 knockout inhibited A431 cell growth. Importantly, miR-6784 was completely ineffective when treating SphK1-knockout A431 cells. Collectively, miR-6784 silences SphK1 and inhibits skin SCC cell progression.

This research introduces an application of an electric impedance sensing technique to investigate cell attachment of normal epithelial cells (HaCAT) and cancerous cells (A431) before and after addition of Ganoderma Lucidum (reishi). In this study, an impedance sensing system is used to measure and characterize real-time changes in electric impedance (resistance and capacitance) with respect to an alternating current (AC) applied to HaCAT and A431 cell colonies. The impedance data is related to the properties of cell spreading, attachment, and delamination. The effect of reishi at dosages of 0.005, 0.01, and 0.02 mg/ml on these cellular properties was inferred from impedance data. The initial impedance data show that resistance is greater for A431 cells than HaCAT cells and that capacitance for A431 cells is less than the capacitance for HaCAT cells. Further, the data shows the resistance for HaCAT cells and for A431 cells increases with time, and the capacitance for both decreases with time. The impedance data analysis shows that reishi plays no role in altering the impedance of the cellular matrix. At most, reishi serves as a lubricant to allow partial detachment and reattachment of HaCAT cell-to-cell bonds, thus reordering (< entropy) the cellular matrix. This effect is not seen in A431 cell colonies.

Wang Zheng,1 Zong-Yu Li,1 De-Lai Zhao,1,2 Xing-Long Li,1,2 Rui Liu3 1Department of Burns, The Fifth Hospital of Harbin, Harbin 150040, People’s Republic of China; 2Department of Orthopedic Surgery, The Fifth Hospital of Harbin, Harbin 150040, People’s Republic of China; 3Department of Burns, Heilongjiang Provincial Hospital, Harbin 150036, People’s Republic of ChinaCorrespondence: Rui LiuDepartment of Burns, Heilongjiang Provincial Hospital, No. 82, Zhongshan Road, Xiangfang District, Harbin 150036, People’s Republic of ChinaTel +86-13268968547Email liurui435342@163.comPurpose: To investigate the specific effect and underlying mechanism of microRNA-26a-5p (miR-26a) in cutaneous squamous cell carcinoma (CSCC).Methods: miR-26a and MMP14/16 mRNA expression were detected by qRT-PCR analysis. Functional experiments were used to detect the role of miR-26a on CSCC progression. Western blot was used for protein detection. Luciferase assay was used to detect miR-26a directly targeting MMP14 and MMP16. Xenograft nude mice model was used to determine the effect of miR-26a on tumorigenesis.Results: miR-26a was decreased in CSCC tissues and cells. Forced miR-26a suppressed the progression of SCL-1 and A431 cells. Furthermore, miR-26a directly targeted MMP14 and MMP16 to inhibit their expression. Forced expression of MMP14 and MMP16 removed the miR-26a’s inhibitory effect on CSCC development. The in vivo tumor growth assay showed that miR-26a suppressed CSCC tumorigenesis by targeting MMP14 and MMP16.Conclusion: Our study suggested miR-26a inhibits cancer cell proliferation, migration and invasion in CSCC by targeting MMP14 and MMP16.Keywords: miR-26a, cutaneous squamous cell carcinoma, MMP14, MMP16, tumorigenesis

Dysregulation of microRNAs (miRNAs) is acknowledged in human cutaneous squamous cell carcinoma (cSCC). We hereby evaluated the ability of miRNA-216b (miR-216b) to impact human cSCC. cSCC tissues with corresponding adjacent normal tissues were collected from 40 patients diagnosed with cSCC where the expression pattern of miR-216b and targeting protein for Xenopus kinesin-like protein 2 (TPX2) was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot analysis. A431 cells were transfected with miR-216b mimic, miR-216b inhibitor, or short interfering RNA against TPX2 to evaluate cell proliferation, invasion, migration, and apoptosis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, scratch test, Transwell assay, and flow cytometry. TPX2 was highly expressed in cSCC tissues while miR-216b was poorly expressed in association with tumor differentiation, lymph node metastasis, and tumor node metastasis staging in patients with cSCC. In response to overexpressed miR-216b or silenced TPX2, cSCC cell proliferation, invasion, and migration were suppressed and apoptosis was stimulated, along with activated p53 signaling. Thus, upregulated miR-216b was capable of promoting apoptosis and inhibiting proliferation, invasion, and migration of cSCC cells by downregulating TPX2 through activation of the p53 signaling, highlighting a novel biomarker for novel treatment modalities against cSCC.

meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second-generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e., Foscan, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g., biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24, and 45 nm) based on benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) (PCLn-PEG; n = 9, 15, or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt % using a film-hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that overexpress the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR-targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR-targeted micelles at 4 °C to EGFR-overexpressing A431 cells, compared to low-EGFR-expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC-loaded micelles by A431 cells was observed when these were decorated with the EGa1 nanobody, compared to nontargeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC loaded in EGa1-conjugated PCL23-PEG (EGa1-P23) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P23 micelles also showed selective PDT against A431 cells compared to the low-EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after intravenous injection, mTHPC incorporated in the P23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.

Selective isolation of circulating tumor cells (CTCs) from blood provides a non-invasive avenue for the diagnosis, prognosis and personalized treatment for patients with cancer. The specific capture of CTCs is conventionally based on the immunoaffinity recognition between antibody and receptor on cell membranes. However, using a traditional antibody for high-efficiency isolation of CTCs remains a challenge due to the limited loading capacity of the large antibodies on material surfaces. Herein, using a small-sized nanobody (Nb), we developed a widely applicable strategy to construct reversible site-specifically immobilized Nb surfaces for the capture and release of epidermoid cancer cell line A431 cells. Coordination interaction between the histidine tag (His-tag) of the nanobody (Nb) and Ni2+ ions that chelated to the NTA-modified poly(2-hydroxyethyl methacrylate) (PHEMA) brushes was used to achieve site-specific immobilization of EGFR Nb (PHEMA-aEGFR surfaces). The high-density immobilized nanobody possessing maximized activity resulted in the high-efficiency capture of 81% rare A431 cells within just 30 min, showing a higher capture yield and shorter capture time compared with that achieved by the conventional antibody immobilized on the flat surface. Additionally, the PHEMA-aEGFR surfaces exhibited low capture limit (1 cell mL-1), cytocompatibility for captured cells, as well as negligible non-specific adhesion of PBMCs. With a one-step treatment using imidazole for competitive coordination, 86% of the captured cells were effectively released. This multifunctional and dynamic site-specifically immobilized nanobody strategy paves a new path in the development of materials and instruments for the high-efficiency capture and release of rare cells at a low cost.

© The Author(s) 2021., In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6β4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the β4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces β4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of β4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds β4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of β4 or reconstituted with phenylalanine specific mutants of β4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated β4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells., This work was supported by grants from the Netherlands Organization for Scientifc Research (NWO; Project Number 824.14.010) and the Dutch Cancer Society (Project Number 2013-5971). JdP acknowledges the Spanish Ministry of Science and Innovation (MCINN), Agencia Estatal de Investigación, and the European Regional Development Fund (ERDF) (Grant PID2019-105763 GB-I00).

Simple Summary Photodynamic therapy (PDT) is used for the treatment of in situ cutaneous squamous cell carcinoma (cSCC), the second most common form of skin cancer, as well as for its precancerous form, actinic keratosis. However, relapses after the treatment can occur. Transforming growth factor β1 (TGFβ1) produced by cancer-associated fibroblasts (CAFs) in the tumor microenvironment has been pointed as a key player in the development of cSCC resistance to other therapies, such as chemotherapy. Here, we demonstrate that TGFβ1 produced by CAFs isolated from patients with cSCC can drive resistance to PDT in SCC cells. This finding opens up novel possibilities for strategy optimization in the field of cSCC resistance to PDT and highlights CAF-derived TGFβ1 as a potential target to improve the efficacy of PDT. Abstract As an important component of tumor microenvironment, cancer-associated fibroblasts (CAFs) have lately gained prominence owing to their crucial role in the resistance to therapies. Photodynamic therapy (PDT) stands out as a successful therapeutic strategy to treat cutaneous squamous cell carcinoma. In this study, we demonstrate that the transforming growth factor β1 (TGFβ1) cytokine secreted by CAFs isolated from patients with SCC can drive resistance to PDT in epithelial SCC cells. To this end, CAFs obtained from patients with in situ cSCC were firstly characterized based on the expression levels of paramount markers as well as the levels of TGFβ1 secreted to the extracellular environment. On a step forward, two established human cSCC cell lines (A431 and SCC13) were pre-treated with conditioned medium obtained from the selected CAF cultures. The CAF-derived conditioned medium effectively induced resistance to PDT in A431 cells through a reduction in the cell proliferation rate. This resistance effect was recapitulated by treating with recombinant TGFβ1 and abolished by using the SB525334 TGFβ1 receptor inhibitor, providing robust evidence of the role of TGFβ1 secreted by CAFs in the development of resistance to PDT in this cell line. Conversely, higher levels of recombinant TGFβ1 were needed to reduce cell proliferation in SCC13 cells, and no induction of resistance to PDT was observed in this cell line in response to CAF-derived conditioned medium. Interestingly, we probed that the comparatively higher intrinsic resistance to PDT of SCC13 cells was mediated by the elevated levels of TGFβ1 secreted by this cell line. Our results point at this feature as a promising biomarker to predict both the suitability of PDT and the chances to optimize the treatment by targeting CAF-derived TGFβ1 in the road to a more personalized treatment of particular cSCC tumors.

A novel quinazoline series of photoswitchable combretastatin A-4 (CA-4) analogues were synthesized and their photochemical properties and antiproliferative activity against A431 epidermoid carcinoma cells were studied. It was found that quinazoline analogues, in contrast to the majority of the known CA-4, exhibit high antiproliferative activity in the E-form as well. Photoswitching of the E-form to the Z-form resulted in a multiple (9-fold) increase in antiproliferative activity. 1H NMR monitoring showed that these compounds are very resistant to UV (λ = 365 nm) or sunlight irradiation and do not undergo photodegradation with a loss of antiproliferative activity that is inherent in heterocyclic analogues of CA-4. Similar photoswitching and an increase in antiproliferative activity are observed on exposure to sunlight. A selected compound (1a-Z51) in sub-micromolar concentrations induced apoptosis in A431 cells, while rad50/ATM/p53 were not involved in cell death. The growth of A431 cells was significantly inhibited after combination treatment with compound 1a-Z51 and chemotherapy drugs (cisplatin or 5-fluorouracil). In summary, the quinazoline analogues of CA-4 represent a promising strategy to achieve a photoswitchable potency for the treatment of cancers, including the development of combination therapies.

MicroRNAs (miRs) have been demonstrated to be associated with multiple processes in the development and progression of human malignancies. Previous studies have observed aberrant downregulation of miR‑144 in several types of cancer, including osteosarcoma. However, the function of miR‑144 and the underlying mechanism in osteosarcoma remain to be elucidated. The present study indicated that miR‑144 was markedly downregulated in osteosarcoma tissues and cell lines compared with that in the normal controls. Restoration of miR‑144 significantly inhibited cell proliferation, migration and invasion of MG‑63 osteosarcoma cells. In addition, Rho‑associated coiled‑coil containing protein kinase 1 (ROCK1) was identified as a novel target of miR‑144 in MG‑63 osteosarcoma cells. Furthermore, knockdown of ROCK1 suppressed the proliferation, migration and invasion of MG‑63 osteosarcoma cells to a similar extent to the effects of miR‑144 overexpression. In addition, the mRNA expression of ROCK1 was increased in osteosarcoma tissues and was negatively correlated with the expression of miR‑144. In conclusion, the results of the present study suggested that miR‑144 acts as a tumor suppressor by targeting ROCK1 in osteosarcoma.

MicroRNA (miR)-138 generally has a suppressive role in various human cancer types; however, its role and the underlying mechanisms in laryngeal squamous cell carcinoma (LSCC) have remained to be elucidated. The present study assessed the clinical significance and regulatory mechanisms of miR-138 in LSCC progression. Reverse-transcription quantitative polymerase chain reaction analysis indicated that miR-138 was significantly downregulated in LSCC tissues and cell lines. In addition, the decreased expression of miR-138 was significantly associated with poor differentiation, lymph node metastasis and advanced clinical stage of LSCC. Restoration of miR-138 expression caused a significant decrease in the proliferation of Hep-2 LSCC cells, while knockdown of miR-138 significantly promoted Hep-2 cell proliferation. A luciferase reporter assay further identified enhancer of zeste homologue 2 (EZH2) as a direct target gene of miR-138, and the protein expression of EZH2 was negatively regulated by miR-138 in Hep-2 cells. Furthermore, overexpression of EZH2 eliminated the suppressive effects of miR-138 on Hep-2 cell proliferation via activation of phosphoinositide-3 kinase (PI3K)/AKT signaling. In addition, EZH2 was found to be significantly upregulated in LSCC tissues and to be inversely correlated to the miR-138 levels. The results of the present study demonstrated that miR-138 inhibits the proliferation of LSCC cells, at least partly via targeting EZH2 and inhibiting PI3 K/AKT signaling. The present study highlighted the clinical significance of the miR-138/EZH2 axis in LSCC.

MicroRNA (miR), a class of small non-coding RNA, function as key regulators in gene expression through directly binding to the 3' untranslated region of their target mRNA, which further leads to translational repression or mRNA degradation. miR-19a, a member of miR-17-92 cluster, has an oncogenic role in a variety of malignant tumors. However, the exact role of miR-19a in nasopharyngeal carcinoma (NPC) has not previously been studied. The present study aimed to investigate the function and mechanism of miR-19a in regulating the viability and invasion of NPC cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) data indicated that the expression levels of miR-17-92 cluster members (miR-17, miR-18a, miR-19a and miR-20a) were frequently increased in NPC tissues compared to the normal tissues. It was also demonstrated that miR-19a was significantly upregulated in NPC C666-1 cells compared to NP69 cells (P

Cancer cells reprogram their metabolism to survive and grow. Small-molecule inhibitors targeting cancer are useful for studying its metabolic pathways and functions and for developing anticancer drugs. Here, we discovered that glutipyran and its derivatives inhibit glycolytic activity and cell growth in human pancreatic cancer cells. According to proteomic profiling of glutipyran-treated cells using our ChemProteoBase, glutipyran was clustered within the group of endoplasmic reticulum (ER) stress inducers that included glycolysis inhibitors. Glutipyran inhibited glucose uptake and suppressed the growth of various cancer cells, including A431 cells that express glucose transporter class I (GLUT1) and DLD-1 GLUT1 knockout cells. When cotreated with the mitochondrial respiration inhibitor metformin, glutipyran exhibited a synergistic antiproliferative effect. Metabolome analysis revealed that glutipyran markedly decreased most metabolites of the glycolytic pathway and the pentose phosphate pathway. Glutipyran significantly suppressed tumor growth in a xenograft mouse model of pancreatic cancer. These results suggest that glutipyran acts as a broad-spectrum GLUT inhibitor and reduces cancer cell growth.

Previous studies have confirmed that the gap junction protein Connexin26 (Cx26) is specifically expressed in human skin tissue. Cx26 can transmit radiation-induced damage signals. However, no study has yet reported whether Cx26 expression affects the radiosensitivity of human skin squamous cancer cells or the mechanism by which this occurs. In this study, we found that human skin squamous cell carcinoma cells (A431 cells) expressed significantly more Cx26 and were more sensitive to radiation compared to normal human keratinocytes (HaCaT cells). Knockdown of Cx26 in A431 cells (A431Cx26–/–) decreased radiosensitivity relative to control cells and altered the expression of key proteins in the MAPK and NF-κB signaling pathways. These results demonstrate that Cx26 expression might play an important role in mediating radiation damage in A431 cells and could serve as a potential target for clinical radiotherapy for cutaneous squamous cell carcinoma.

The present study showed that the recombinant analogue of SLURP‑1 effectively inhibits the growth of a 3D model of tumors - multicellular spheroids from human epidermoid carcinoma A431 cells and human lung adenocarcinoma A549 cells. The combined application of SLURP‑1 with gefitinib (inhibitor of epidermal growth factor receptor (EGFR)) leads to the synergistic antiproliferative effect on spheroids from A431 cells. The results obtained suggest the possibility for design of first-in-class anticancer drugs based on recombinant SLURP‑1.

Objective. This study is aimed at investigating the effects of Lycium barbarum polysaccharide (LBP) on the proliferation and apoptosis of human cutaneous squamous cell carcinoma A431 cells in vitro and in vivo via its regulation on autophagy. Methods. In vitro experiment: A431 cells were treated with different concentrations of LBP, and cell viability was measured by the CCK8 method. Flow cytometry was used to detect the cell apoptosis rate. The expression of Ki67, PCNA, cl-caspase-3, Bcl-2, and LC3II and the phosphorylation status of JNK and ERK1/2, as well as the effect of SP600125 cotreatment on the expression of autophagy and apoptosis-associated proteins, were determined via Western blot. In vivo experiment: a transplanted tumor model was established by subcutaneous injection of A431 cells to the nude mice. 50 mg/kg LBP was injected into the mice intraperitoneally; the survival rate of mice, volume, and weight of tumor were determined on the 30th day. The expression of Ki67 and MMP-2 proteins was measured by immunohistochemistry. Results. LBP at concentrations of 400 μg/ml and above was significantly cytotoxic to A431 cells, whereas, within the dose range of 50 μg/ml~200 μg/ml, LBP significantly inhibited the expression of Ki67 and PCNA proteins, promoted the expression of cl-caspase-3, inhibited the expression of Bcl-2 protein, downregulated the expression of autophagy marker LC3II, and reduced the phosphorylation of ERK1/2, whereas the level of JNK phosphorylation was upregulated. At the same time, the regulation of Beclin1, LC3II, Bcl-2, and cl-caspase-3 by LBP was effectively reversed by the cotreatment of SP600125. In addition, LBP increased the survival rate of transplanted nude mice, reduced tumor volume and weight, and downregulated the expression of Ki67 and MMP-2. Conclusion. LBP can induce apoptosis of A431 cells by inhibiting autophagy and can inhibit tumor growth in vivo.

In the present study we showed that the recombinant analogue of the SLURP-1 protein effectively inhibits the growth of a 3D model of tumors-multicellular spheroids reconstructed from human epidermoid carcinoma A431 cells and human lung adenocarcinoma A549 cells. The combined application of rSLURP-1 with gefitinib (inhibitor of epidermal growth factor receptor (EGFR)) leads to the synergistic antiproliferative effect on spheroids from A431 cells. The results obtained suggest the possibility for design of first-in-class anticancer drugs based on recombinant SLURP-1.

Drug-drug interactions (DDIs), especially with herbal medicines, are complex, making it difficult to identify potential molecular mechanisms and targets. We introduce a workflow to carry out DDI research using transcriptome analysis and interactions of a complex herbal mixture, Compound Kushen Injection (CKI), with cancer chemotherapy drugs, as a proof of principle. Using CKI combined with doxorubicin or 5-Fu on cancer cells as a model, we found that CKI enhanced the cytotoxic effects of doxorubicin on A431 cells while protecting MDA-MB-231 cells treated with 5-Fu. We generated and analysed transcriptome data from cells treated with single treatments or combined treatments and our analysis showed that opposite directions of regulation for pathways related to DNA synthesis and metabolism which appeared to be the main reason for different effects of CKI when used in combination with chemotherapy drugs. We also found that pathways related to organic biosynthetic and metabolic processes might be potential targets for CKI when interacting with doxorubicin and 5-Fu. Through co-expression analysis correlated with phenotype results, we selected the MYD88 gene as a candidate major regulator for validation as a proof of concept for our approach. Inhibition of MYD88 reduced antagonistic cytotoxic effects between CKI and 5-Fu, indicating that MYD88 is an important gene in the DDI mechanism between CKI and chemotherapy drugs. These findings demonstrate that our pipeline is effective for the application of transcriptome analysis to the study of DDIs in order to identify candidate mechanisms and potential targets.

Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop.Methods: The 286–303 peptide was coupled chemically to keyhole limpet hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V regions were cloned and sequenced. Receptor binding was characterized using enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines.Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be cytotoxic to MDA-MB-468, A431 and F98npEGFRvIII expressing cells.Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

Purpose: This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453). Results: Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization. Conclusion: This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.

Background Survivin, a member of the inhibitor of apoptosis protein family, is highly expressed in many cancers and has important roles in inhibiting apoptosis by blocking caspase activation. However, its antitumor effects remain largely unknown. Here we explore the function of survivin in skin cancer. Methods We used qPCR and Western blot to examine survivin expression in skin cancer patients and cell line. We generated several survivin shRNA constructs and tested the effects of survivin shRNA on cancer cell viability using MTT assay, flow cytometry, and TUNEL assay. Results We found that survivin was upregulated in both skin cancer patients and skin cancer cell line A431. Knockdown survivin via shRNA inhibited cancer cell proliferation and promoted apoptosis in both A431 cell and in vivo xenograft tumor mouse model. The antitumor effect is comparable to resveratrol, a drug known to inhibit cancer progression. Moreover, we showed that inhibition of survivin was able to increase the expression of cleaved caspase 7/caspase 9 and activate the ataxia-telangiectasia mutated-NF-κB pathway in A431 cells. Conclusion Survivin-shRNA possesses antitumor abilities in vitro and in vivo by inhibiting the proliferation and promoting apoptosis of A431 cells. It may serve as a potential anticancer target for skin cancer therapy in the future.

Detecting the presence of circulating tumor cells (CTCs) in peripheral blood can be useful for monitoring treatment in patients, metastasis prognosis, and even early detection. The epidermal growth factor receptor (EGFR) is overexpressed in carcinoma, e.g. in colorectal cancer. Here, we use atomic force microscopy (AFM) force spectroscopy to study the mechanical properties of A431 cells, which simulate EGFR-overexpressing epithelial CTCs and were magnetically isolated by Bio-Ferrography (BF). BF is found useful in isolating individual cancerous cells for mechanical testing, thus avoiding cell-cell interactions. Different stages in the pre-isolation sample preparation steps (namely, cell fixation, PLL coating of the glass substrate, and immunomagnetic labeling) are found to affect the estimated Young's modulus. The BF magnetic isolation step itself does not change the elasticity of the captured cells in comparison to the pre-isolated microbeads-bound cells. The reported increase in the estimated Young's modulus between BF-isolated target cells and fixed cells that are not bound to magnetic microbeads can be used as a quantitative mechanical indicator for objective detection of CTCs. Furthermore, we report a 2.8-fold increase in the adhesion force between the AFM tip and the BF-isolated cells compared to the pre-isolated magnetic microbead-bound A431 fixed cells. This adhesion force correlation could potentially serve as an additional quantitative mechanical indicator for distinguishing between the target and background cells, without the use of cell staining assay and subjective analysis by an expert pathologist. This study demonstrates the powerful combination of the highly sensitive cell isolation by BF and the subsequent analysis of mechanical properties of individual captured cancerous cells by AFM. This combination has potential use in cancer research.

This study was aimed at evaluating the role of bifunctional chelators DOTA-NCS and CHX-A″-DTPA-NCS used for conjugating 177 Lu with Nimotuzumab on the radiochemical yields, purity, in vitro stability, and specificity of the radioimmunoconjugates to EGFR. Two immunoconjugates were prepared wherein Nimotuzumab was conjugated with the acyclic ligand p-NCS-Bn-CHX-A″-DTPA and macrocyclic ligand p-NCS-Bn-DOTA. These were radiolabeled with 177 Lu, purified on PD-10 column, and characterized by SE-HPLC. In vitro stability was determined up to 4 days post preparation. Specificity of the radioimmunoconjugates was ascertained by in vitro studies in A431 cells while the biodistribution patterns were studied in normal Swiss mice up to 96 hours post injection. Four to five molecules of CHX-A″-DTPA/DOTA were attached to one molecule of Nimotuzumab. Radiochemical purity of both 177 Lu-CHX-A″-DTPA-Nimotuzumab and 177 Lu-DOTA-Nimotuzumab was determined to be greater than 98%. Both the radioimmunoconjugates exhibited good in vitro stability at 37°C up to 4 days post preparation in saline, and their clearance was largely by the hepatobiliary route. The DOTA- and CHX-A″-DTPA-based radioimmunoconjugates could be prepared with good radiochemical purity, in vitro stability, and specificity to EGFR. Further studies in EGFR-positive cancers would pave way for them for use in the clinics.

AIM: This study aimed at developing antibody-functionalized gold nanoparticles (AuNPs) to selectively target cancer cells and probing their potential radiosensitizing effects under proton irradiation.MATERIALS & METHODS: AuNPs were conjugated with cetuximab (Ctxb-AuNPs). Ctxb-AuNP uptake was evaluated by transmission electron microscopy and atomic absorption spectroscopy. Radioenhancing effect was assessed using conventional clonogenic assay.RESULTS & CONCLUSION: Ctxb-AuNPs specifically bound to and accumulated in EGFR-overexpressing A431 cells, compared with EGFR-negative MDA-MB-453 cells. Ctxb-AuNPs enhanced the effect of proton irradiation in A431 cells but not in MDA-MB-453 cells. These data indicate, for the first time, that combining enhanced uptake by specific targeting and radioenhancing effect, using conjugated AuNPs, is a promising strategy to increase cell killing by protontherapy.

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.

BACKGROUND Radix Tetrastigma Hemsleyani Flavone (RTHF) has detoxification and anti- inflammation activity and is widely used. Here, we report that RTHF inhibits cell proliferation and induces apoptosis in cutaneous squamous cell carcinoma A431 cells and is a potential strategy for cancer therapy. MATERIAL AND METHODS A431 cells were cultured in different concentrations of RTHF. The inhibition of cell proliferation was assessed by MTT assay, cell apoptosis was shown through FCM, and cell invasion was assessed by Transwell methods. Enzyme proteasome assay was used to detect the activity of proteasome and DUB. Expression of apoptosis-related and ubiquitin proteasome pathway-associated proteins were assessed by PCR and Western blot. RESULTS RTHF obviously suppressed the proliferation and induced apoptosis of A431 cells in a dose-dependent manner. Transwell assay showed that RTHF inhibited the cell metastasis significantly. Enzyme proteasome assay show that the RTHF treatment of activity of proteasome and DUB was significantly lower than in control. RTHF increased the expression of Bax and inhibited Bcl-2, pro-caspase3, and pro-caspase9 activity. The expression of USP14, UCHL5, and POH1 decreased and ub-prs increased significantly in the treatment group. CONCLUSIONS Our study reveals that RTHF-mediated inhibition of DUBs and proteasome may provide a potential strategy for cancer therapy.

miRNA has shown its potential in the regulation of squamous cell carcinoma (SCC). However, the mechanism of such an effect was not quite clear. Therefore, we aimed to investigate whether miR-30a-5p participated in the regulation of cutaneous SCC (cSCC) and the possible mechanism involved. 5-Ethynyl-2'-deoxyuridine (EdU) and cell cycle were measured using flow cytometry. The formation of cell colony was tested by colony formation assay. The capacities of migration and invasion were tested by wound healing assay and Transwell invasion assay, respectively. The target of miR-30a-5p was predicted by bioinformatics and identified by luciferase assay. Western blot was used for the determination of proteins and qPCR was for mRNA levels. miR-30a-5p expression was lowered in SCL-1 and A431 cells, and its upregulation suppressed EdU positive cells, colony numbers, migration, invasion and Bcl-2 expression, and elevated Bcl-2-associated X protein (Bax) and cleaved Caspase-3 expressions, arresting cell cycle in G1 phase. Moreover, forkhead box protein G1 (FOXG1) was proved to be the target of miR-30a-5p, and FOXG1 overexpression partially offsets the decreased colony numbers, migration and invasion rates due to miR-30a-5p overexpression in SCL-1 and A431 cells. miR-30a-5p showed a regulatory role on the expression of FOXG1 and further modulated the progressing of cSCC cells, which could be a novel pathway intervening the development of cSCC.

Background: Cutaneous squamous cell carcinoma (CSCC) is the most deadly disease among nonmelanoma skin cancers. LINC00641 plays a role in various cancers, but its role in CSCC has not been reported...

The performance of quasi-spherical gold nanoparticles (GNPs) on the generation of reactive oxygen species (ROS) to cause cell damage, as irradiated by a two-photon laser, is studied. In this mechanism, hot electrons are generated from GNPs as irradiated by the two-photon laser, reacting with the molecules in the medium to produce ROS. We used laser scanning confocal microscopy with a low-fluence femtosecond Ti:Sapphire laser of 800 nm to observe the generated ROS in A431 cells, which were incubated with GNPs in advance. Subsequently, the cell morphology, cytoskeleton, and viability were investigated. In comparison with the control (no GNPs), the expression of ROS in these GNP-treated cells was enhanced after irradiation by the two-photon laser. Additionally, the disruption of cytoskeletons and the follow-up apoptosis of these GNP-treated cells are significantly increased as the number of laser shots increases. Moreover, we used N-acetyl-L-cysteine (NAC), an antioxidant, to inhibit the formation of ROS, to clarify whether the cytoskeletal disruption is caused by ROS rather than photothermal effects. Our results show that after two-photon irradiation, the ROS expression in these cells treated with GNPs plus NAC was significantly reduced. In addition, the cytoskeletal damage of these cells treated with GNPs and NAC was less than that of those treated with GNPs but without NAC, their cell viability after three days was almost the same with the control. These results illustrate that the induced ROS from the two-photon excited GNPs is the main cause of cell damage. The study may pave a way for the use of GNPs as a photosensitized therapeutic agent for two-photon photodynamic therapy on tumor treatment.

Background and aim Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. Methods ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. Results In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-μM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 μM and ZnPCS4 at 2.5 μM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. Conclusions Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 μM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1–10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03042-y.

Simple Summary Variable domains of heavy chain only antibodies are small proteins that can be used for tumor imaging and therapy upon conjugation of functional groups. As frequently used random conjugation techniques can decrease binding to the target of interest, site-specific conjugation of these functional groups is preferred. Here, we optimized site-specific conjugation of both a chelator for binding of a radiometal and a photosensitizer to epidermal growth factor receptor (EGFR) binding VHH 7D12. We characterized this dual-labeled VHH for nuclear imaging and targeted photodynamic therapy of EGFR-expressing tumors. Abstract Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. 111In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.

Purpose To investigate the safety of photobiomodulation therapy (PBM) in tumors and its potential as a radiosensitizer when combined with radiotherapy. Methods We have performed in vitro experiments in A431 cells to assess proliferation and cell cycle after PBM, as well as clonogenic assay and H2AX-gamma immunolabeling to quantify double strand breaks after the combination of PBM and radiation. In vivo experiments in xenografts included Kaplan-Meier survival analysis, optical coherence tomography (OCT) and histological analysis. Results PBM did not induce proliferation in vitro, but increased the G2/M fraction by 27% 24h after illumination, resulting in an enhancement of 30% in radiation effect in the clonogenic assay. The median survival of the PBM-RT group increased by 4 days and the hazard ratio was 0.417 (CI 95%: 0.173–1.006) when compared to radiation alone. OCT analysis over time demonstrated that PBM increases tumor necrosis due to radiation, and histological analysis showed that illumination increased cell differentiation and angiogenesis, which may play a role in the synergetic effect of PBM and radiation. Conclusion PBM technique may be one of the most appropriate approaches for radiosensitizing tumors while protecting normal tissue because of its low cost and low training requirements for staff.

Combined 5-fluorouracil (5-FU) and melittin (MEL) is believed to enhance cytotoxic effects on skin squamous cell carcinoma (SCC). However, the rationale underlying cytotoxicity is fundamentally important for a proper design of combination chemotherapy, and to provide translational insights for future therapeutics in the dermatology field. The aim was to elucidate the effects of 5-FU/MEL combination on the viability, proliferation and key structures of human squamous cell carcinoma (A431). Morphology, plasma membrane, DNA, mitochondria, oxidative stress, cell viability, proliferation and cell death pathways were targeted for investigation by microscopy, MTT, trypan blue assay, flow cytometry and real-time cell analysis. 5-FU/MEL (0.25 µM/0.52 µM) enhanced the cytotoxic effect in A431 cells (74.46%, p < .001) after 72 h exposure, showing greater cytotoxic effect when compared to each isolated compound (45.55% 5-FU and 61.78% MEL). The results suggest that MEL induces plasma membrane alterations that culminate in a loss of integrity at subsequent times, sensitizing the cell to 5-FU action. DNA fragmentation, S and G2/M arrest, disruption of mitochondrial metabolism, and alterations in cell morphology culminated in proliferation blockage and apoptosis. 5-FU/MEL combination design optimizes the cytotoxic effects of each drug at lower concentrations, which may represent an innovative strategy for SCC therapy.

Transforming growth factor-β (TGF-β) has an important role in multiple target genes and signaling pathways. The E2F family of transcription factors is a group of DNA-binding proteins that are involved in cell-cycle progression, and therefore have a key role in proliferation. The present study demonstrates that inhibition of cell growth by TGF-β occurs in the multiple myeloma cell line MM.1S. However, the growth-suppressive effects of TGF-β may be reversed by small interfering (si)RNA to reduce the expression of E2F1. TGF-β1 and E2F1 siRNA were manipulated in MM.1S cells to investigate the association between these genes. FACScan Flow Cytometer, western blot analysis and other methods were adopted to confirm such interrelation. The present data showed that TGF-β mediated growth suppression in MM.1S cells, while inducing E2F1 protein expression levels rapidly and transiently. The present data support the hypothesis that E2F1 is a central mediator of TGF-β-induced growth suppression in MM.1S cells and control of E2F1 may be a downstream event of TGF-β action, at least in one multiple myeloma cell line.

Skin cancer is the deadliest type of malignant disease and causes primary mortality worldwide. Dioscin, which exists in medicinal plants, has potent anticancer effects. However, its effects on skin cancer remain unknown. In the present study, the activity and mechanism of dioscin on the human skin cancer A431 cell line were investigated, MTT, colony formation, Transwell, wound-healing, TUNEL, Comet, immunofluorescence and western blot assays were used to assess the effects of dioscin on A431 cells. The results of MTT, colony formation, Transwell and wound-healing assays revealed that dioscin suppressed proliferation, colony formation and invasion of the cancer cells. TUNEL and comet assays demonstrated that dioscin exhibited significant effects on cell apoptosis and DNA damage. Investigations into the mechanism revealed that the expression levels of phosphorylated Ataxia telangiectasia-mutated (ATM) were considerably activated by dioscin, which significantly upregulated the expression levels of p53 to activate mitochondrial apoptosis signaling. Furthermore, the expression levels of BAX, cleaved caspase-3/9 and cleaved poly (ADP-ribose) polymerase were upregulated, and the expression levels of BCL-2 were downregulated by dioscin. Additionally, dioscin markedly downregulated the expression levels of matrix metalloproteinase 2 (MMP2), MMP9, RHO and cdc42, which are all associated with tumor invasion. In addition, p53-small interfering RNA transfection experiments indicated that dioscin exhibited excellent activity against skin cancer in vitro by decreasing p53 expression. Overall, the present results suggested that dioscin inhibited skin cancer cell proliferation via adjusting ATM/p53-mediated cell apoptosis, migration and DNA damage, which should be considered as a potential option for future treatments of skin cancer.

Although long non‑coding RNAs (lncRNAs) have been implicated in various human cancer types, the role of lncRNA ezrin antisense RNA 1 (EZR‑AS1) in cutaneous squamous cell carcinoma (cSCC) remains unclear. The present study aimed to investigate the effect of lncRNAEZR‑AS1 on cSCC and identify the underlying molecular mechanisms. EZR‑AS1 expression was measured in cSCC tissue and cells detected using reverse transcription‑quantitative PCR. Gain‑of‑function assays were performed in A431 cells, which have a relatively low expression of EZR‑AS1, while loss‑of‑function assays were performed in SCC13 and SCL‑1 colon cancer cells, which have a relatively high expression of EZR‑AS1. Cell viability, proliferation, migration, invasion and apoptosis were assessed using MTT, plate cloning, wound healing, Transwell and flow cytometry assays, respectively. EZR‑AS1 mRNA expression levels were significantly upregulated in cSCC tissues and cells compared with adjacent healthy tissues and HaCaT cells, respectively. Compared with the small interfering RNA (si)‑negative control (NC) group, si‑EZR‑AS1 significantly inhibited SCC13 and SCL‑1 cell proliferation, migration and invasion, but promoted cell apoptosis. By contrast, compared with the pc‑NC group, EZR‑AS1 overexpression significantly enhanced A431 cell proliferation, migration and invasion, but inhibited cell apoptosis. Moreover, focal adhesion kinase (FAK) was identified as a target of EZR‑AS1, and EZR‑AS1 knockdown significantly decreased FAK expression compared with the si‑NC group. Moreover, EZR‑AS1 knockdown significantly downregulated the protein expression levels of phosphorylated (p)‑PI3K/PI3K and p‑AKT/AKT in cSCC cells compared with the si‑NC group. The PI3K agonist 740Y‑P significantly reversed si‑EZR‑AS1‑mediated effects on SCC13 and SCL‑1 cell proliferation, migration, invasion and apoptosis. In conclusion, the present study demonstrated that si‑EZR‑AS1 inhibited cSCC cell proliferation, migration and invasion, and promoted cell apoptosis, potentially via regulating the PI3K/AKT signaling pathway. Therefore, the present study provided novel insights into the diagnosis and treatment of cSCC.

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, which can progress rapidly and has a poor prognosis. Glypican-3 (GPC3) has been proposed to be an important diagnostic biomarker and therapeutic target for HCC. Aptamers have emerged as promising drug delivery vehicles because of their high binding affinity for target molecules. Herein, we developed G12msi, a gemcitabine-incorporated DNA aptamer, targeting GPC3, and evaluated its binding specificity and anti-tumor efficacy in GPC3-overexpressing HCC cell lines and murine xenograft models. GPC3-targeted aptamers were selected by using the SELEX process and the chemotherapy drug gemcitabine was internally incorporated into the aptamer. To determine the binding affinity and internalization of the G12msi, flow cytometry and confocal microscopy were performed on GPC3-positive HepG2, Hep3B, and Huh7 cells, as well as a GPC3-negative A431 cell. The anti-tumor activities of G12msi were evaluated with in vitro and in vivo models. We found that G12msi binds to GPC3-overexpressing HCC tumor cells with high specificity and is effectively internalized. Moreover, G12msi treatment inhibited the cell proliferation of GPC3-positive HCC cell lines with minimal cytotoxicity in control A431 cells. In vivo systemic administration of G12msi significantly inhibited tumor growth of HCC HepG2 cells in xenograft models without causing toxicity. These results suggest that gemcitabine-incorporated GPC3 aptamer-based drug delivery may be a promising strategy for the treatment of HCC.

Conjugation of growth factors to a carrier is a favorable method to improve their efficacy as therapeutic molecules. Here, we report the carrier size effect on bioactivity of human epidermal growth factor (hEGF) conjugated to polystyrene particles. BALB/3T3 cells were treated with hEGF-conjugated particles (hEGF-conjs) sized from 20 to 1000 nm. At hEGF concentrations less than 0.5 ng ml-1, free hEGF was more potent than the hEGF-conjs at inducing cell proliferation. However, cell proliferation was size-dependent at higher concentrations of hEGF i.e. hEGF-conjs sized equal to or less than 200 nm displayed lower cell proliferation, compared to free hEGF, but larger particles showed increased cell proliferation. This is in agreement with previous studies showing accumulation of activated-EGFRs in early endosomes triggers apoptosis of A431 and HeLa cells. The confocal microscopy and co-localization fluorescence staining showed the 500 and 1000 nm hEGF-conjs exclusively remained on the cell surface, probably enabling them to activate EGF receptors for a longer time. Conversely, smaller particles were mostly inside the cells, indicating their rapid endocytosis. Similarly, A431 cells treated with 20 nm hEGF-conj, endocytosed the particles and experienced decreased cell proliferation, while the 500 and 1000 nm hEGF-conjs were not internalized, and induced partial cell proliferation. Moreover, we showed multivalency of hEGF-conjs is not the cause of enhanced cell proliferation by large particles, as the degree of EGFR phosphorylation by free EGF was higher, compared to hEGF-conjs. Our results suggest the potential of micron-sized particles as a carrier for hEGF to enhance cell proliferation, which could be explored as a promising approach for topical application of growth factors for accelerating wound healing.

Administration of cetuximab (C-mab) in combination with paclitaxel (PTX) has been used for patients with head and neck squamous cell carcinoma (SCC) clinically. In this study, we attempted to clarify the molecular mechanisms of the enhancing anticancer effect of C-mab combined with PTX on oral SCC cells in vitro. We used two oral SCC cells (HSC4, OSC19) and A431 cells. PTX alone inhibited cell growth in all cells in a concentration-dependent manner. C-mab alone inhibited the growth of A431 and OSC19 cells at low concentrations, but inhibited the growth of HSC4 cells very weakly, even at high concentrations. A combined effect of the two drugs was moderate on A431 cells, but slight on HSC4 and OSC19 cells. A low concentration of PTX enhanced the antibody-dependent cellular cytotoxicity (ADCC) induced by C-mab in all of the cells tested. PTX slightly enhanced the anticancer effect of C-mab in this ADCC model on A431 and HSC4 cells, and markedly enhanced the anticancer effect of C-mab on OSC19 cells. These results indicated that PTX potentiated the anticancer effect of C-mab through enhancing the ADCC in oral SCC cells.

The synthesis of both enantiomers of 3-[(tert-butyldimethylsilyl)oxy]methyl-4,5-O-isopropylidenecyclopent-2-en-1-ol was accomplished in six steps based on optically inactive dimethyl meso-tartrate. This key intermediate in the synthesis of cyclopentenyl carbocyclic nucleosides was subsequently applied in the preparation of enantiomeric neplanocins A. The toxic effect of these compounds was investigated for a series of suspension and adherent cancer cell lines and normal human fibroblasts. (−)-Neplanocin A ((−)-NPA) was more toxic against all tested cancer cell lines than its dextrorotary counterpart. The highest toxicity with IC50 values of 7 and 10 μM was observed for the MOLT-4 and A431 cells, respectively. Moreover, (−)-NPA also induced apoptosis in A431 cell while this effect was not observed for (+)-NPA.

BACKGROUND Cutaneous squamous cell carcinoma (cSCC) is the second most common cutaneous malignancy with an incidence rate increasing each year. Glutathione S-transferase A3 (GSTA3), a member of the glutathione S-transferase family, is considered an antioxidative protease, but its role in cSCC remains unclear. AIM The present study was designed to explore the effect of GSTA3 on cSCC. PATIENTS/METHODS Through previous systematic studies, we screened GSTA3 to be a key gene with lower expression in cSCC. In the present study, we selected cSCC tissues and para-carcinoma tissue specimens from 20 patients in plastic surgery department. A431 cells were treated with GSTA3 transfection. The cell proliferation, apoptosis, colony formation, and cell migration as well as invasion were examined, respectively. And the expressions of GSTA3, TGF-β/Smad, and HIF-1α signalings were measured by Western blot and qRT-PCR. RESULTS GSTA3 was downregulated in both cSCC tissues and A431 cells. Additionally, overexpression of GSTA3 induced a phenotype with a lower degree of malignancy, while GSTA3 silencing induced more malignant phenotypes, including cell proliferation, colony formation, apoptosis, migration, and invasion. Moreover, we found that the TGF-β/Smad2/3 and HIF-1α signalings were activated in cSCC under hypoxic conditions. CONCLUSION GSTA3 could inhibit cSCC progression through suppression of the TGF-β/Smad and HIF-1α signalings. Therefore, GSTA3 may prove to be a prospective therapeutic target for cSCC.

BackgroundSmall cell lung cancer (SCLC) accounts for 15% of lung cancers, and the primary treatment of this malignancy is chemotherapy and radiotherapy. Delta-like 3 (DLL3) is an attractive target for SCLC immunotherapy since its expression is highly restricted to SCLC with a neglectable appearance on normal adult tissues. In the current study, we aimed to explore the efficacy of DLL3-targeted SCLC immunotherapy via the engagement of T cell.MethodsAs a proof of concept, we constructed DLL3-targeted bispecific antibody and chimeric antigen receptor (CAR)-modified T cells. In vitro and in vivo tumor-suppression activity of these treatments alone or in combination with a Program Death-1 (PD-1) inhibitory antibody was evaluated.ResultsIn vitro studies showed that both DLL3 bispecific antibody and CAR-T efficiently killed DLL3-positive cancer cells, including the native SCLC cell lines H446, H196, H82, and the artificial A431 cells that were forcefully overexpressing DLL3. In vivo studies in xenograft mouse models demonstrated that both bispecific antibody and CAR-T suppressed the tumor growth, and combination therapy with PD-1 inhibitory antibody dramatically improved the efficacy of the DLL3 bispecific antibody, but not the CAR-T cells.ConclusionsOur results demonstrated that DLL3-targeted bispecific antibody plus PD-1 inhibition was effective in controlling SCLC growth.

Targeted delivery of anticancer drugs that selectively accumulate in malignant cells could enhance drug efficacy and reduce side effects of conventional chemotherapy. In this work, we designed a single domain antibody (nanobody) based drug delivery system for targeted delivery of anticancer drugs. An anti-EGFR nanobody (Nb) was constructed with a C3-tag and a Q-tag for site specific modifications under physiological conditions. The site specific PEGylation of the nanobody was achieved via a transglutaminase catalyzed reaction through the coupling of the Q-tag with PEG-NH2. As a proof of concept, the PEGylated nanobody was tethered to HSA coated upconversion nanoparticles (UCNPs) through the C3-tag, and an anticancer drug, doxorubicin (DOX), was loaded. Results showed that the Nb-conjugated drug delivery system exhibits superior specificity to the EGFR positive tumor cells. The drug delivery system is highly accumulated in the EGFR positive tumor cells (A431), whereas there was no detectable accumulation in the EGFR negative cells (MCF-7). Consequently, the drug loaded particles demonstrated significantly higher anti-proliferation to A431 cells than to MCF-7 cells. This work provides an effective approach for site-specific modification of nanobodies for the construction of targeted drug delivery systems.