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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.

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.

Cancer metastasis and recurrence are potentially lethal. A small number of cancer cell groups called cancer stem cells (CSCs) have both stem cell capacity and cancer-forming ability and are reported to play important roles in cancer metastasis and recurrence. These CSCs are considered to be radiation-resistant (RR). Therefore, understanding the biological effects of radiation on squamous cell carcinoma (SCC) cell lines in vitro and in vivo might be worthwhile to circumvent radiation resistance. Currently, there are no reports on the establishment of RR-SCC cells in serum-free defined culture, which mimics biological mechanisms and prevents instability of using serum in the culture medium. We isolated radiation-resistant strains, designated A431-LDR and A431-HDR, from A431 cells derived from vulval SCC and irradiated them with a total dose of 60 Gy at a low-dose rate (2.2 Gy/d) (RM1000) and a high-dose rate (5 Gy/5.75min) in serum-free defined culture. These cells exhibited high sphere-forming and migration ability in vitro and high tumor-forming ability in nude mice xenografts. Overexpression of KRT13 in A431-RR cells might play a role in its radiation-resistant characteristics. These cells might be useful not only to study cancer stem cells but also to study the circumvention of radiation resistance by novel cancer treatment modalities.

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.

Chemical modification of the thiol group on protein tyrosine phosphatase (PTP) 1B triggers an activation of epidermal growth factor receptor (EGFR) signaling that is mimicked by environmental electrophiles through S-modification of PTP1B. While activation of PTP1B/EGFR by a single exposure to an electrophile has been established, the effects of combined exposure to electrophiles are unknown. Here, we examined the activation of EGFR signaling by combined exposure to ambient electrophiles in human epithelial carcinoma A431 cells. Simultaneous exposure to 1,2- and 1,4-naphthoquinone (NQ) augmented the S-modification of endogenous and recombinant human PTP1B (hPTP1B). Combined exposure of hPTP1B or A431 cells to 1,2- and 1,4-NQ escalated the inactivation of PTP compared with individual exposure. Phosphorylation of EGFR and its downstream kinase extracellular signal-regulated kinase (ERK) 1/2 by 1,2-NQ exposure was facilitated by simultaneous exposure to 1,2-NQ with 10 µM 1,4-NQ. An EGFR inhibitor diminished the phosphorylation of ERK1/2, indicating that ERK was phosphorylated following EGFR activation by the NQ cocktail. The combined exposure to NQs also accelerated cell death in A431 cells compared with each NQ alone. While no EGFR/ERK activation was seen following 1,4-benzoquinone (BQ) treatment, exposure to 1,4-NQ in the presence of 1,4-BQ increased 1,4-NQ-mediated activation of EGFR. This suggests that the enhancement of 1,4-NQ-dependent EGFR activation by 1,4-BQ is caused by a different mechanism than 1,2-NQ with 1,4-NQ. These results suggest that combined exposure to ambient electrophiles, even at low concentrations, can induce stronger activation of redox signaling than individual exposure. Our findings indicate that combining different electrophiles may produce unexpected effects.

Background Basal cell carcinoma (BCC) is the most common form of skin carcinoma. Aim To investigate the function of key micro(mi)RNAs and to explore the potential molecular mechanisms involved in BCC. Methods The microarray dataset GSE34535, which comprises seven BCC samples and seven control samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) were identified. We collected tissue samples from 20 patients with BCC and 20 healthy controls (HCs), to compare the miR-18a expression in their tissue samples. Expression of miR-18a in A431 and HaCaT cells was also assayed. Following this, we upregulated and downregulated miR-18a expression in A431 cells to examine the effects on cell proliferation, migration and apoptosis. To further investigate the relative mechanism, the proteins LC3, Beclin 1, Akt and mammalian target of rapamycin (mTOR) were examined by quantitative real-time PCR and Western blotting. For further verification, we examined the expression of LC3 in the 20 BCC and 20 HC tissue samples. Results In total, 19 DE-miRNAs (13 upregulated and 6 downregulated) that were common to the BCC and HC groups were identified. Levels of miR-18a were about three-fold higher in BCC tissues and A431 cells compared with their respective control groups. In vitro, downregulation of miR-18a was shown to inhibit cell proliferation and activate autophagy via the Akt/mTOR signalling pathway, while upregulation of miR-18a promoted proliferation of these cells. LC3 was decreased in BCC compared with HC tissue samples. Conclusions Our data support an oncogenic role of miR-18a through a novel Akt/mTOR/Beclin 1/LC3 axis, and suggest that the antitumour effects of miR-18a inhibitor may make it suitable for BCC therapy.

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.

Objective The SAM- and SH3-domain containing 1 gene (SASH1) has been considered as a tumor suppressor in some cancers. Nevertheless, the effect of SASH1 on the proliferation and invasion of human skin squamous cell carcinoma (cSCC) remains poorly understood. Therefore, the purpose of the present study was to observe the potential role of SASH1 in cSCC and investigate its underlying mechanisms. Methods The overexpression of SASH1 was constructed by transfecting the pcDNA3.1/SASH1 vector into SCL-1 and A431 cells, and SASH1 knockdown was generated by transfecting the SASH1 siRNA into cSCC cells. Then, cell proliferation, invasion, apoptosis, and Akt pathway were observed. Results The expression levels of SASH1 mRNA and protein were greatly reduced in cSCC cells. The overexpression of SASH1 inhibited the viability and invasion of cSCC cells, while its knockdown induced the viability and invasion of cSCC cells. The overexpression of SASH1 also suppressed the expression levels of p-Akt and its target genes, including cyclin D1, Bcl-2, and metal matrix proteinase 2(MMP-2). By contrast, SASH1 knockdown exerted the opposite role. Furthermore, inhibition of Akt obviously decreased the inducible effect of cSCC knockdown on the proliferation and invasion of cSCC cells. Conclusion Overall, these results found that SASH1 inhibits the proliferation and invasion of cSCC cells via suppressing Akt cascade, indicating a tumor inhibitory effect of SASH1 in cSCC cells.

Purpose To evaluate the anti-tumor effects of oxymatrine in vulvar squamous cell carcinoma (VSCC) cells and to explore the underlying mechanisms. Methods We selected SW962 and A431 VSCC cell lines. Cell proliferation was examined using MTT assay. Cell cycle and apoptosis were detected using flow cytometry. Migration and invasion were evaluated using transwell and wound-healing assays. The relevant protein expression and signaling pathways were analyzed using Western blotting. Results Oxymatrine inhibited the proliferation of SW962 and A431 VSCC cells in a time- and dose-dependent manner. Oxymatrine induced cell cycle arrest in the G2/M phase by increasing the protein expression of P21 and decreasing levels of cyclin B1 and CDC2. Oxymatrine upregulated the expression of cleaved-caspase 3 and BAX and downregulated the expression of BCL2, which led to an increase in apoptosis. Oxymatrine also suppressed the migration and invasion of SW962 and A431 cells by reducing levels of MMP2 and MMP9. After treatment with oxymatrine or a RAS inhibitor (salirasib), expression levels of RAS, p-RAF, p-MEK, p-ERK, C-MYC, and MMP2 were reduced. When TGF-β1 was used to stimulate SW962 and A431 cells, the expression of the above proteins increased; this increase was reversed by using oxymatrine or salirasib again. Conclusion Oxymatrine inhibits proliferation and migration of VSCC cells by blocking the RAS/RAF/MEK/ERK pathway.

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

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.

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.

Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell–cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

Cutaneous squamous cell carcinoma (CSCC) is the second most common skin cancer. Dihydromyricetin (DHM), a Rattan tea extract, has been shown to have antitumor activity with no obvious toxicity to normal cells in vitro and in vivo. However, its efficacy in the treatment of CSCC and the underlying antitumor mechanism has not been fully elucidated yet. In our study, DHM increased autophagic flux in the A431 cells, as evidenced by the upregulation of LC3-II and downregulation of P62/SQSTM1. Moreover, the pharmacological or genetic blocking autophagy decreased DHM-induced cell death, indicating DHM triggered autophagic cell death in A431 cells. Specifically, DHM induced TFEB(Ser142) de-phosphorylation, activated TFEB nuclear translocation and increased of TFEB reporter activity, which contributed to the expression of autophagy-related genes and subsequent initiated autophagic cell death in A431 cells. Importantly, DHM decreased lncRNA MALAT1 expression and MALAT1 overexpression abrogated the effects of DHM on TFEB-dependent autophagy both in vitro and in vivo. Taken together, DHM induces CSCC cell death via inducing excessive autophagy, which is mediated through the MALAT1-TFEB pathway. Therefore, DHM may be beneficial for the development of chemotherapy for CSCC.

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.

Neuroblastoma breakpoint family member 1 (NBPF1) is involved in the occurrence and development of tumors. However, only a limited number of studies were conducted on NBPF1 and cutaneous squamous cell carcinoma (SCC). This study mainly explored the expression and mechanism of NBPF1 in SCC. SCC tissue and adjacent tissues samples were randomly selected. NBPF1 gene was overexpressed in the A431 cell line using plasmid transfection technique. Cell viability was tested by cell counting kit-8 (CCK-8) assay. Flow cytometry was used to determine cell cycle and apoptosis. Western blot and RT-qPCR were respectively performed to determine the expression levels of proteins and mRNAs. The NBPF1 gene was lowly expressed in SCC tissues. The expression level of NBPF1 gene was the lowest in A431 cell line. The cell viability of A431 was reduced after transfection. Overexpression of NBPF1 not only arrested A431 cells in G1 phase and promoted apoptosis, but also up-regulated the expressions of Bax and p53 mRNA and protein and down-regulated the expressions of Bcl-2, Survivin and Cyclin D1. Akt-p53-Cyclin pathway was inhibited when NBPF1 gene expression was up-regulated. Upregulation of NBPF1 might promote apoptosis of A431 cells and block cell cycle via inhibiting the activation of Akt-p53-Cyclin signaling pathway.

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...

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, and its incidence is rising because of the aging population. Nectin cell adhesion molecule 4 (NECTIN4) is involved in the progression of tumors and has attracted interest as a potential therapeutic target. However, little is known about the expression and significance of NECTIN4 in cSCC. The aim of this study was to determine the expression and function of NECTIN4 in cSCC. Immunohistological NECTIN4 expression was investigated in tissues from 34 cSCC patients. Using an A431 human SCC cell line, the role of NECTIN4 in the regulation of cell–cell attachment and migration and proliferation was assessed. NECTIN4 was expressed in most cSCC tissues and on the plasma membrane of A431 cells. Silencing of NECTIN4 prevented cell–cell attachment and induced the expression migration-related molecules, leading to an increase in cell migration. Knockdown of NECTIN4 downregulated extracellular signal-regulated kinase signaling, decreased cyclin D1 expression, and inhibited cell proliferation. These results show that NECTIN4 is expressed in cSCC and functions in the regulation of cell–cell interactions, as well as in the migration and proliferation of SCC cells. NECTIN4-targeted therapy may serve as a novel and promising treatment for cSCC.

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.

Costunolide is a naturally occurring sesquiterpene lactone that demonstrates various therapeutic actions such as anti-oxidative, anti-inflammatory, and anti-cancer properties. Costunolide has recently emerged as a potential anti-cancer agent in various types of cancer, including colon, lung, and breast cancer. However, its mode of action in skin cancer remains unclear. To determine the anti-cancer potential of costunolide in skin cancer, human epidermoid carcinoma cell line A431 was treated with costunolide. A lactate dehydrogenase assay showed that costunolide diminished the viability of A431 cells. Apoptotic cells were detected by annexin V/propidium iodide double staining and Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay assay, and costunolide induced cell apoptosis via activation of caspase-3 as well as induction of poly-ADP ribose polymerase cleavage in A431 cells. In addition, costunolide elevated the level of the pro-apoptotic protein Bax while lowering the levels of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. To address the inhibitory effect of costunolide on cell proliferation and survival, various signaling pathways, including mitogen-activated protein kinases, signal transducer and activator of transcription 3 (STAT3), nuclear factor κB (NF-κB), and Akt, were investigated. Costunolide activated the p38 and c-Jun N-terminal kinase pathways while suppressing the extracellular signal-regulated kinase (ERK), STAT3, NF-κB, and Akt pathways in A431 cells. Consequently, it was inferred that costunolide suppresses cell proliferation and survival via these signaling pathways. Taken together, our data clearly indicated that costunolide exerts anti-cancer activity in A431 cells by suppressing cell growth via inhibition of proliferation and promotion of apoptosis. Therefore, it may be employed as a potentially tumor-specific candidate in skin cancer treatment.

An important mediator of tumorigenesis, the epidermal growth factor receptor (EGFR) is expressed in almost all non-transformed cell types, associated with tumor progression, angiogenesis and metastasis. The significance of the EGFR as a cancer therapeutic target is underscored by the clinical development of several different classes of EGFR antagonists, including monoclonal antibodies (mAb) and tyrosine kinase inhibitors. Extensive preclinical studies have demonstrated the anti-tumor effects of mAb806 against tumor xenografts overexpressing EGFR. EGF stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signalling proteins which regulate cell proliferation and apoptosis. Detailed understanding of the intracellular signalling pathways and components modulated by mAbs (such as mAb806) to EGFR, and other growth factor receptors, remain limited. The use of fluorescence 2D difference gel electrophoresis (2D DIGE), coupled with sensitive MS-based protein profiling in A431 tumor (epidermoid carcinoma) xenografts, in combination with mAb806, revealed proteins modulating endocytosis, cell architecture, apoptosis, cell signalling pathways and cell cycle regulation, including Dynamin-1-like protein, cofilin-1 protein, and 14-3-3 protein zeta/delta. Further, we report various proteins, including Interferon-induced protein 53 (IFI53), and Oncogene EMS1 (EMS1) which have roles in the tumor microenvironment, regulating cancer cell invasiveness, angiogenesis and formation of metastases. These findings contribute to understanding the underlying biological processes associated with mAb806 therapy of EGFR-positive tumors, and identifying further potential protein markers that may contribute in assessment of the treatment response. © 2013 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted.

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.

Cutaneous squamous-cell carcinoma (cSCC) is one of most common type of non-black skin cancer. The malignancy degree and the death risk of cSCC patients are significantly higher than basal cell carcinoma patients. GL-V9 is a synthesized flavonoid derived from natural active ingredient wogonin and shows potent growth inhibitory effects in liver and breast cancer cells. In this study, we investigated the anti-cSCC effect and the underlying mechanism of GL-V9. The results showed that GL-V9 induced both apoptosis and autophagy in human cSCC cell line A431 cells, and prevented the growth progression of chemical induced primary skin cancer in mice. Metabolomics assay showed that GL-V9 potentially affected mitochondrial function, inhibiting glucose metabolism and Warburg effect. Further mechanism studies demonstrated that AKT played important roles in the anti-cSCC effect of GL-V9. On one hand, GL-V9 suppressed AKT-modulated mitochondrial localization of HK2 and promoted the protein degradation of HK2, resulting in cell apoptosis and glycolytic inhibition. On the other hand, GL-V9 induced autophagy via inhibiting Akt/mTOR pathway. Interestingly, though the autophagy induced by GL-V9 potentially antagonized its effect of apoptosis induction, the anti-cSCC effect of GL-V9 was not diluted. All above, our studies suggest that GL-V9 is a potent candidate for cSCC treatment.

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.

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.

Bromodomain-containing protein 4 (BRD4) is a potential therapeutic target of skin squamous cell carcinoma (SCC). I-BET726 is a novel BRD4 inhibitor. Its potential effect in skin SCC cells was tested in the present study. We show that I-BET726 potently inhibited survival, proliferation, cell cycle progression, and migration in established (A431/SCC-9/SCC-12/SCC-13 lines) and primary human skin SCC cells. I-BET726 induced significant apoptosis activation in skin SCC cells. It was more efficient in inhibiting skin SCC cells than known BRD4 inhibitors (JQ1, CPI203, and AZD5153). I-BET726 not only downregulated BRD4-regulated proteins (c-Myc, Bcl-2, and cyclin D1), but also inhibited sphingosine kinase 1 (SphK1) and Akt signalings in SCC cells. Restoring Akt activation, by a constitutively active S473D mutant Akt1 (“caAkt1”), partially inhibited I-BET726-induced cytotoxicity in A431 cells. In vivo, I-BET726 oral administration potently inhibited A431 xenograft growth in severe combined immunodeficient mice. Downregulation of BRD4-regulated proteins and inhibition of the SphK1-Akt signaling were detected in I-BET726-treated A431 xenograft tumor tissues. Together, I-BET726 inhibits skin SCC cell growth in vitro and in vivo.

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.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathophysiological functions. Also, HB-EGF plays a pivotal role in progression of different tumors. So, HB-EGF seems to be a target molecule for the treatment of some cancer types. Aim To obtain HB-EGF neutralizing polyclonal antibodies and test their anti-proliferative properties in vitro. Materials and methods Lab rabbits and mice were used for immunization with recombinant HB-EGF. The effect of generated polyclonal antibodies on viability and apoptosis of human epidermoid carcinoma derived A431 cell line was assessed using MTT and Annexin V-propidium iodide assays. Results Rabbit polyclonal anti-HB-EGF serum could block binding of soluble HB-EGF to epidermal growth factor receptor/human epidermal growth factor receptor. Also, anti-HB-EGF antibodies could bind to surface of A431 cells which express abnormally high levels of membrane bound proHB-EGF and its receptor. It has been shown that immune serum with polyclonal antibodies against HB-EGF was able to block the mitogenic activation of the cells with HB-EGF and cause apoptotic cell death. Conclusion Inhibition of HB-EGF activity with neutralizing polyclonal antibodies can effectively inhibit mitogenic activation and cause apoptosis of cancer cells with significant epidermal growth factor receptor overexpression.

Heparin-binding protein 17/fibroblast growth factor–binding protein-1 (HBp17/FGFBP-1) was purified from A431 cell-conditioned media based on its capacity to bind to fibroblast growth factor 1 and 2 (FGF-1 and FGF-2). HBp17/FGFBP-1 has been observed to induce the tumorigenic potential of epithelial cells and is highly expressed in oral cancer cell lines and tissues. HBp17/FGFBP-1 is also recognized as a pro-angiogenic molecule as a consequence of its interaction with FGF-2. We have previously reported that Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, downregulated the expression of HBp17/FGFBP-1 and inhibited the proliferation of squamous cell carcinoma (SCC) cells in vitro and in vivo through NF-κb inhibition. To explore the possibility of microRNA (miRNA) control of HBp17/FGFBP-1, we analyzed exosomal miRNAs from medium conditioned by A431 cells treated with ED-71. Microarray analysis revealed that 12 exosomal miRNAs were upregulated in ED-71-treated A431 cells. Among them, miR-6887-5p was identified to have a predicted mRNA target matching the 3′ untranslated region (3′-UTR) of HBp17/FGFBP-1. The 3′-UTR of HBp17/FGFBP-1 was confirmed to be a direct target of miR-6887-5p in SCC/OSCC cells, as assessed with a luciferase reporter assay. Functional assessment revealed that overexpression of miR-6887-5p in SCC/OSCC cells inhibited cell proliferation and colony formation in vitro, and inhibited tumor growth in vivo compared with control. In conclusion, our present study supports a novel anti-cancer mechanism involving the regulation of HBp17/FGFBP-1 function by exosomal miR-6887-5p in SCC/OSCC cells, which has potential utility as a miRNA-based cancer therapy., This research was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology to T.O. (grant number: 15H05043) and T. S. (grant number: 16K11723).

Squamous cell carcinoma (SCC) is a usually responds poorly to treatment suffers from poor therapeutic benefits while oroxylin A (OA) is a promising flavonoid with high anticancer efficacy against various cancer types. Here in our study, in order to reveal the potential of OA based drug delivery systems (DDSs) in the treatment of SCC, we firstly revealed that OA had a certain pharmacodynamic effect on skin SCC (A431 cells). Afterwards, OA was loaded into a newly synthesized aggregation-induced emission (AIE)-active polymer to construct OA-loaded PDots for the first time. Our results revealed that OA-loaded PDots showed preferable drug loading and enhanced stability. Moreover, the DDS was also capable of self-illumination in the aggregate state to reveal the uptake profile. Most importantly, the DDS showed much more elevated anticancer benefits than free OA in vitro and advanced tumor targetability in vivo, suggesting that it might be a promising system against SCC.

Epidermal growth factor receptor (EGFR) is overexpressed in a wide range of solid tumors. In this study, we exploited a high-affinity EGFR-antagonistic affibody (ZEGFR) coupled to a doxorubicin loaded pegylated liposome (LS-Dox) for concurrent passive and active targeting of EGFR+ A431 tumor cells in vitro and in vivo. The in vitro studies revealed that the Dox liposomes coupled with ZEGFR (AS-Dox) showed a higher Dox uptake than LS-Dox in EGFR+ A431 cells but not in EGFR- B16F10 cells, resulting in a selectively enhanced cytotoxicity. In vivo, AS-Dox confirmed its long circulation time and efficient accumulation in tumors. This targeted chemotherapy achieved greater tumor suppression. Further, this low-dose but effective targeted treatment reduced systemic toxicity such as body weight loss and organ injury demonstrated by H&E staining. Thus, selective targeting of LS-Dox coupled with ZEGFR enhanced antitumor effects and improved systemic safety. These results demonstrated that LS-Dox coupled with ZEGFR might be developed as a potential tool for therapy of EGFR+ tumors.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging., Theranostics, 10 (24), ISSN:1838-7640

There have been many studies on improving the efficacy of cisplatin and on identifying safe compounds that can overcome multi-drug resistance (MDR) acquired by cancer cells. Our previous research showed that polyethylene glycol-modified titanium dioxide nanoparticles (TiO2 PEG NPs) affect cell membrane receptors, resulting in their aggregation, altered localization and downregulation. TiO2 PEG NPs may affect P-glycoprotein (P-gp), a membrane efflux channel involved in MDR. In this study, we investigated the effect of TiO2 PEG NPs on cisplatin cytotoxicity. We used HepG2 cells, which highly express P-gp and A431 cells, which show low expression of P-gp. The results showed that 10 &micro, g/mL 100 nm TiO2 PEG NPs increased intracellular cisplatin levels and cytotoxicity in HepG2 cells but not in A431 cells. TiO2 PEG NPs treatment decreased the expression level of P-gp in HepG2 cells. Our findings indicate that TiO2 PEG NPs enhance cisplatin cytotoxicity by down regulating P-gp and that TiO2 PEG NPs are promising candidates for inhibiting P-gp and reversing drug resistance acquired by cancer cells.