Radix Tetrastigma Hemsleyani Flavone represses cutaneous squamous cell carcinoma via Janus kinase/signal transducer and activator of transcription 3 pathway inactivation.

Cutaneous squamous cell carcinoma (CSCC) is the second most common malignant skin tumor and significantly affects patients' quality of life and health. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway activation is involved in CSCC development. Radix Tetrastigma hemsleyani flavone (RTHF) is an active Radix Tetrastigma extract (RTE), which was recently reported to have promising inhibitory effects on CSCC. However, the underlying functional mechanisms of this inhibition remain unknown. In the present study, A431 cells or SCL-1 cells were incubated with 1, 5, and 10 mg/mL RTHF for 48 h, respectively. A significantly increased wound closure rate, decreased number of migrated and invaded cells, decreased colony number, and elevated apoptotic rate were observed after treatment with 1, 5, and 10 mg/mL RTHF. Furthermore, after incubation with RTHF, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 levels were drastically reduced. An A431 xenograft model was constructed, followed by oral administration of 15, 30, or 60 mg/kg RTHF for 21 consecutive days. A significantly lower increase in tumor volume and reduced tumor weight were observed in all RTHF-treated groups. In addition, JAK/STAT3 signaling was drastically repressed in tumor tissues. Collectively, RTHF inhibited CSCC progression, which may be associated with JAK/STAT3 pathway inactivation.

Effects of photodynamic therapy using Red LED-light combined with hypocrellin B on apoptotic signaling in cutaneous squamous cell carcinoma A431 cells.

BACKGROUND: The incidence of cutaneous squamous cell carcinoma (cSCC) has been demonstrating yearly increases. cSCC is a malignant cancer and exerts a major impact on patients' health and quality of life. Thus, the development and use of novel therapies in the treatment of cSCC are needed. It has been reported that LED photodynamic therapy (LED PDT) mediated by Hypocrellin B and its derivatives, a second-generation photosensitizer, can induce apoptosis in a variety of tumor cells, However, its potential pro-apoptotic effects on cSCC have yet to be investigated. OBJECTIVE: This study aims to investigate the pro-apoptotic effects and molecular mechanisms of HB-LED PDT in cutaneous squamous cell carcinoma A431 cells (Subsequent abbreviation A431 cells). Such information can provide an important theoretical foundation for the clinical translation of HB-LED PDT in the treatment of cSCC. METHODS: 1. Effects of HB on A431 cells were determined using a Cell Counting Kit-8 assay, which method can indirectly reflect the number of living cells. In this way, this assay can then provide a means to identify the optimal concentrations of HB required for the induction of apoptosis in A431 cells. 2. The effects of HB-LED PDT on the morphology of A431 cells and changes in the nuclei after Hoechst33342 staining as determined using inverted fluorescent microscopy. 3. Use of the Annexin V-FITC test kit to detect levels of apoptosis in A431 cells in response to treatment with HB. Changes in reactive oxygen species and mitochondrial membrane potential following HB-LED PDT treatment in A431 cells were determined using fluorescence activated cell sorting (FACS). 4. Real-time quantitative PCR and Western Blot were applied to assess changes in several key factors involved in apoptosis including Bax, Bcl-2, and Caspase-3, at both transcription and translation levels. With these assays, it was possible to investigate the apoptotic signaling pathway in A431 cells in response to HB-LED PDT. RESULTS: HB-LED PDT inhibited proliferation activity and promoted nuclear fragmentation within these A431 cells. HB-LED PDT inhibited mitochondrial activity, increased reactive oxygen species production, and promoted apoptosis of A431 cells. In addition, several key factors in the apoptotic signaling pathway were increased at both the transcriptional and translational levels in A431 cells in response to the HB-LED PDT, indicating that the apoptotic signaling pathway was activated by HB-LED PDT. CONCLUSION: HB-LED PDT induces apoptosis in A431 cells through a mitochondria-mediated apoptotic pathway. Such findings serve as an important foundation for the development of new approaches in the treatment of cSCC.

Antitumor effects of erlotinib in combination with berberine in A431 cells.

BACKGROUND: First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, have been shown to target tumors with L858R (exon 21) and exon 19 deletions, resulting in significant clinical benefits. However, acquired resistance often occurs due to EGFR mutations. Therefore, novel therapeutic strategies for treatment of patients with EGFR-positive tumors are needed. Berberine (BBR) is an active alkaloid extracted from pharmaceutical plants such as Coptis chinensis. Berberine has been shown to significantly inhibit EGFR activity and mediate anticancer effects in multiple preclinical studies. We investigated whether combining BBR with erlotinib could augment erlotinib-induced cell growth inhibition of EGFR-positive cells in a mouse xenograft model. METHODS: We examined the antitumor activities and potential mechanisms of erlotinib in combination with berberine in vitro and in vivo using the MTT assay, immunoblotting, flow cytometry, and tumor xenograft models. RESULTS: In vitro studies with A431 cells showed that synergistic cell growth inhibition by the combination of BBR and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, and inhibition of cyclin D and Bcl-2 expression compared to that observed in response to BBR or erlotinib alone. The efficacy of the combination treatment was also investigated in nude mice. Consistent with the in vitro results, BBR plus erlotinib significantly reduced tumor growth. CONCLUSION: Our data supported use of BBR in combination with erlotinib as a novel strategy for treatment of patients with EGFR positive tumors.

Expression of glucose transporter 3 in cutaneous squamous cell carcinoma and its effect on the proliferation, invasion and migration of A431 cells / 中华皮肤科杂志

Objective:To determine the expression of glucose transporter 3 (GLUT3) in cutaneous squamous cell carcinoma (cSCC), and to evaluate its effect on the cSCC cell line A431.Methods:From June 2016 to December 2020, 22 paraffin-embedded tissue specimens were collected from patients with pathologically confirmed cSCC in the Department of Dermatology, Peking University Third Hospital, and 20 discarded normal skin tissues after dermatological surgeries served as controls. Immunohistochemical assay was performed to determine the GLUT3 expression in cSCC tissues and normal skin tissues. Cultured A431 cells were divided into two groups: GLUT3 overexpression group transfected with a lentiviral vector carrying the SLC2A3 gene, and negative control group transfected with an empty lentiviral vector. Real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of GLUT3 in A431 cells in different groups, the cell proliferation assay (MTS assay) was performed to estimate the cell proliferative activity, and the live-cell analysis system Incucyte S3 was used for real-time evaluation of the migratory and invasive abilities of A431 cells in different groups. The relative glucose consumption and lactic acid production in A431 cells at 48 hours were measured by using glucose and lactic acid assay kits, retrospectively. Two independent samples t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. Results:The GLUT3 expression was significantly higher in the cSCC tissues than in the normal skin tissues (immunohistochemical assay score: 9.39 ± 2.56 points vs. 2.30 ± 2.60 points; t = 8.91, P < 0.05). Compared with the negative control group, the mRNA and protein expression of GLUT3 markedly increased in the GLUT3 overexpression group. MTS assay showed significantly increased proliferative activity of A431 cells in the GLUT3 overexpression group compared with the negative control group after 24- and 96-hour treatment ( t = 2.49, 3.54, P = 0.048, 0.012, respectively); cell fusion rates in the scratched area were significantly higher in the GLUT3 overexpression group than in the negative control group in the cell migration assay at 6, 12 18, and 24 hours and cell invasion assay at 12, 18, and 24 hours (all P < 0.05). At 48 hours, the relative glucose consumption and lactic acid production in A431 cells were significantly higher in the GLUT3 overexpression group than in the negative control group ( t = 2.98, 2.20, P = 0.011, 0.038, respectively) . Conclusion:GLUT3 was highly expressed in the cSCC tissues, and may participate in the occurrence and development of cSCC by providing energy to cSCC cells via promoting glucose uptake in cells to enhance their proliferative, migratory and invasive abilities.

Apoptotic mechanism activated by blue light and cisplatinum in cutaneous squamous cell carcinoma cells.

New approaches are being studied for the treatment of skin cancer. It has been reported that light combined with cisplatinum may be effective against skin cancer. In the present study, the effects of specific light radiations and cisplatinum on A431 cutaneous squamous cell carcinoma (cSCC) and HaCaT non­tumorigenic cell lines were investigated. Both cell lines were exposed to blue and red light sources for 3 days prior to cisplatinum treatment. Viability, apoptosis, cell cycle progression and apoptotic­related protein expression levels were investigated. The present results highlighted that combined treatment with blue light and cisplatinum was more effective in reducing cell viability compared with single treatments. Specifically, an increase in the apoptotic rate was observed when the cells were treated with blue light and cisplatinum, as compared to treatment with blue light or cisplatinum alone. Combined treatment with blue light and cisplatinum also caused cell cycle arrest at the S phase. Treatment with cisplatinum following light exposure induced the expression of apoptotic proteins in the A431 and HaCaT cell lines, which tended to follow different apoptotic mechanisms. On the whole, these data indicate that blue light combined with cisplatinum may be a promising treatment for cSCC.

Fabrication of nanopatterned PLGA films of curcumin and TPGS for skin cancer.

Squamous cell carcinoma treatment has limited therapeutic options and the incidence rate is increasing recently. In the present investigation, we developed poly(lactic-co-glycolic acid) (PLGA) nanopatterned films (NPFs) through poly dimethyl siloxane (PDMS) cast molding technique and explored its therapeutic efficacy in combination with curcumin and tocopherol poly (ethylene glycol) 1000 succinate (TPGS). Herein, we demonstrate the preparation and characterization of curcumin loaded tocopherol polyethylene glycol 1000 succinate stabilized poly (lactic-co-glycolic) acid nanopatterned films (CTP-NPFs). CTP-NPFs showed good in vitro cytotoxicity towards human skin cancer cell line (A431) when compared to that of unpattern films. CTP-NPFs effectively inhibited the progression of 7, 12-Dimethylbenz[a]anthracene (DMBA)/croton oil induced skin cancer in Swiss albino mice. The nanopatterned films could be used as an alternate treatment for skin cancer.

Detection and quantification of VEGFR-1 in the nuclei of tumor cells by a new flow cytometry-based method.

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 ß-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.

EGFR-targeting, ß-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant ß-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting ß-defensin consists of an EGF-derived oligopeptide (Ec), a ß-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 µmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting ß-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of ß-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

ESOPE-Equivalent Pulsing Protocols for Calcium Electroporation: An In Vitro Optimization Study on 2 Cancer Cell Models.

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs combined with electroporation represents the new frontier for electrochemotherapy applications; calcium electroporation has been recently proposed as a simple tool for anticancer therapy. In vitro investigations facilitate the optimization of electrical parameters and drugs for in vivo and clinical testing. In this optimization study, new pulsing protocols have been tested by increasing the pulse number and reducing the electric field with respect to the standard. European Standard Operating Procedure on Electrochemotherapy-equivalent protocols have been identified in HL-60 and A431 cancer cell models, and a higher sensitivity in terms of electroporation yield has been recorded in HL-60 cells. Moreover, cell killing efficacy of European Standard Operating Procedure on Electrochemotherapy-equivalent protocols has been demonstrated in the presence of increasing calcium concentrations on both cell lines. Equivalent European Standard Operating Procedure on Electrochemotherapy protocols can be used to optimize the therapeutic effects in the clinic, where different regions of the same cancer tissue, with different electrical properties, might result in a differential electroporation yield of the standard protocol over the same tissue, or, eventually, in an override of the operational limits of the instrument. Moreover, using calcium can help overcome the drawbacks of standard drugs (side effects, high costs, difficult handling, preparation, and storage procedures). These results support the possibility of new treatment options in both standard electrochemotherapy and calcium electroporation, with clear advantages in the clinic.

The effect of near-infrared fluorescence conjugation on the anti-cancer potential of cetuximab.

This study investigated the anti-cancer potential of a near-infrared fluorescence (NIRF) molecule conjugated with Cetuximab (Cetuximab-NIRF) in six-week-old female BALB/c athymic (nu+/nu+) nude mice. A431 cells were cultured and injected into the animals to induce solid tumors. Paclitaxel (30 mg/kg body weight (BW)), Cetuximab (1 mg/kg BW), and Cetuximab-NIRF (0.25, 0.5 and 1.0 mg/kg BW) were intraperitoneally injected twice a week into the A431 cell xenografts of the nude mice. Changes in BW, tumor volume and weight, fat and lean mass, and diameter of the peri-tumoral blood vessel were determined after two weeks. Tumor volumes and weights were significantly decreased in the Cetuximab-NIRF (1 mg/kg BW) group compared with the control group (P<0.001). Lean mass and total body water content were also conspicuously reduced in the Cetuximab-NIRF (1 mg/kg BW) group compared with the vehicle control group. Peri-tumoral blood vessel diameters were very thin in the Cetuximab-NIRF groups compared with those of the paclitaxel group. These results indicate that the conjugation of Cetuximab with NIRF does not affect the anti-cancer potential of Cetuximab and NIRF can be used for molecular imaging in cancer treatments.

Effect of small interfering RNA targeting survivin gene on the expression of survivin and biological function of a human cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.

Effects of turmeric volatile oil combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431 and their mechanisms / 中华皮肤科杂志

Objective To evaluate the effects of turmeric volatile oil (TVO) combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431,and to explore their mechanisms.Methods Some cultured A431 cells at exponential growth phase were divided into several groups to be treated with 5,10,20,40 and 80 mg/L TVO,as well as high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1％ dimethyl sulfoxide (DMSO,control group),respectively.After 24-hour treatment,cell counting kit 8 (CCK8) assay was performed to estimate the proliferative activity of A431 cells in the above groups.Some other A431 cells were divided into 4 groups:control group treated with high-glucose DMEM containing 1％ DMSO,TVO group treated with 40 mg/LTVO,cisplatin group treated with 10 mg/L cisplatin,and TVO + cisplatin group treated with 40 mg/L TVO and 10 mg/L cisplatin.After 24-hour treatment,CCK8 assay was performed to estimate the cellular proliferative activity,inverted microscopy to observe changes in cell morphology,fluorescence microscopy to detect cell apoptosis after acridine orange (AO)/ethidium bromide (EB) double-staining,colorimetry to evaluate the activity of Caspase-3 and Caspase-9,and Western blot analysis to determine the protein expression of Caspase-3 and p-glycoprotein.Results After 24-hour treatment with 5,10,20,40 and 80 mg/L TVO,the cell proliferation rates were inhibited by (12.83 ± 6.4)％,(16.27 ± 11.4)％,(21.61 ± 9.1)％,(33.11 ± 2.0)％ and (46.00 ± 3.3)％ respectively,and the inhibition rates were all significantly higher in these groups than in the control group (4.03％ ± 1.4％,all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of TVO at 24 hours was (61.66 ± 1.03) mg/L.Compared with the control group,the proliferation inhibition rates significantly increased in the TVO group,cisplatin group and TVO + cisplatin group (all P ＜ 0.05),suggesting that the combination of TVO and cisplatin showed synergistic inhibitory effects with a combination index of 1.366.Moreover,A431 cells turned round to different extents and became apoptotic in the TVO group and cisplatin group,and the TVO + cisplatin group showed obviously decreased number of cells and a large number of cell debris.The TVO + cisplatin group also showed significantly increased activity of Caspase-3 (1.520 ± 0.115) and Caspase-9 (2.760 ± 0.297) as well as protein expression of Caspase-3 (1.482 ± 0.016) compared with the TVO group (Caspase-3 activity:1.117 ± 0.095;Caspase-9 activity:1.259 ± 0.059;Caspase-3 protein expression:1.156 ± 0.006,all P ＜ 0.01) and cisplatin group (Caspase-3 activity:1.381 ± 0.089;Caspase-9 activity:1.829 ± 0.171;Caspase-3 protein expression:1.296 ± 0.021,all P ＜ 0.01),but significantly decreased p-glycoprotein expression (0.528 ± 0.014) compared with the TVO group (1.311 ± 0.011,P ＜ 0.01) and cisplatin group (1.169 ± 0.012,P ＜ 0.01).Conclusion TVO combined with cisplatin can synergistically inhibit the proliferation of A431 cells and induce cell apoptosis,which may be associated with activation of the caspase system and decreased expression of pglycoprotein.

The effect of near-infrared fluorescence conjugation on the anti-cancer potential of cetuximab / 한국실험동물학회지

This study investigated the anti-cancer potential of a near-infrared fluorescence (NIRF) molecule conjugated with Cetuximab (Cetuximab-NIRF) in six-week-old female BALB/c athymic (nu+/nu+) nude mice. A431 cells were cultured and injected into the animals to induce solid tumors. Paclitaxel (30 mg/kg body weight (BW)), Cetuximab (1 mg/kg BW), and Cetuximab-NIRF (0.25, 0.5 and 1.0 mg/kg BW) were intraperitoneally injected twice a week into the A431 cell xenografts of the nude mice. Changes in BW, tumor volume and weight, fat and lean mass, and diameter of the peri-tumoral blood vessel were determined after two weeks. Tumor volumes and weights were significantly decreased in the Cetuximab-NIRF (1 mg/kg BW) group compared with the control group (P < 0.001). Lean mass and total body water content were also conspicuously reduced in the Cetuximab-NIRF (1 mg/kg BW) group compared with the vehicle control group. Peri-tumoral blood vessel diameters were very thin in the Cetuximab-NIRF groups compared with those of the paclitaxel group. These results indicate that the conjugation of Cetuximab with NIRF does not affect the anti-cancer potential of Cetuximab and NIRF can be used for molecular imaging in cancer treatments.

HDR brachytherapy decreases proliferation rate and cellular progression of a radioresistant human squamous cell carcinoma in vitro.

PURPOSE: To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters. MATERIALS AND METHODS: An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method. RESULTS: Significant decreasing cellular proliferation rate (p < 0.05) as well as reduced survival fraction (p < 0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G2/M phase (32.3 ± 1.5%) in the irradiated group as compared with untreated cells (8.22 ± 1.2%) was detected. Also, a significant (p < 0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found. CONCLUSION: HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G2/M cell cycle arrest.

EGFR-mediated apoptosis via STAT3.

The Epidermal Growth Factor Receptor (EGFR) is a cell surface receptor with primary implications in cell growth in both normal and malignant tissue. Paradoxically, cell lines that hyperexpress the EGFR have been documented to undergo receptor-mediated apoptosis. The underlying mechanism by which EGF-induced apoptosis occurs however remains inexplicit. In an attempt to identify this mechanism, we assessed downstream effectors of EGFR in MDA-MB-468 cells during conditions of EGF-induced apoptosis. The effector assessment revealed STAT3 as a potential mediator of EGF-induced apoptosis. Alternative strategies for activating STAT3, independent of EGFR stimulation, resulted in the induction of the apoptotic pathways. A reduction in STAT3 expression via RNAi resulted in a significant attenuation of EGF-induced PARP cleavage. Our findings support STAT3 as a positive mediator of EGF-induced apoptosis in MDA-MB-468 cells.

Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis.

Stylosanthes guianensis is a fodder legume native from South America and widely grown worldwide. Dried plant material was purchased on the web and taxonomically identified by light and SEM microscopy, and morphological analysis of plants germinated from seeds. The plant was extracted with dichloromethane:2-propanol (9:1). Bioguided fractionation using calcein-AM cytotoxicity assay on HeLa and A431 tumor cells allowed to isolate a lipophilic fraction, endowed with strong cytotoxicity. By means of 1- and 2-D NMR, HPLC-MS, and HR-ESIMS it could be seen that the fraction was an inseparable mixture of complex lipids, mainly consisting of esterified 3-hydroxy fatty acids. Acidic methanolysis of the mixture yielded 3-OH C10 and C12 carboxylic acids, together with palmitic, stearic, and arachidonic acids. Mass values indicate the presence of dimeric and trimeric combinations of 3-hydroxy, C10/C12 acids, and C16/C18/C20 acids, linked via ester bond. Monomeric hydroxyl-fatty acids were also observed, in particular derivatives of mono-hydroxy and di-hydroxy linolenic, linoleic, and oleic acids. 3-O-acylated, esterified fatty acids are unusual in higher plants, and recall motifs of Gram-negative endotoxin lipid A. These oxylipins are likely to be responsible for the antiproliferative activity of S. guianensis, suggesting possible use of the plant in the development of antitumor drugs.

Proteomic Analysis and Functional Studies of Baicalin on Proteins Associated with Skin Cancer.

Abundant evidence supports the key role of ultraviolet radiation (UVR) in skin cancer development. The human skin, especially the epidermal layer, is the main defense against UV radiation. Baicalin is a major bioactive component of Scutellaria baicalensis Georgi, a plant which has been found to exhibit antitumor activity. The anticarcinogenic mechanism of baicalin is not completely understood. We have reported that baicalin inhibited UVB-induced photo-damage and apoptosis in HaCaT cells (human skin keratinocytes). The aim of the present study is to investigate the cellular gene targets responsible for baicalin's antitumor activity by performing two-dimensional electrophoresis liquid chromatography-mass spectrometry/mass spectrometry (2-DE LC-MS/MS) with HaCaT cells following UVB and baicalin exposure. Two-DE for protein separation was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches. Nucleophosmin (NPM)-specific siRNA was designed and synthesized, and the small interfering RNA was transfected into skin squamous cancer A431 cells to knockdown the NPM expression. Proliferation and cell cycle status were assessed by CCK8 and flow cytometric analyses, respectively. We have identified 38 protein spots that are differentially expressed in HaCaT cells exposed to baicalin and/or UVB irradiation These proteins are involved in detoxification, proliferation, metabolism, cytoskeleton and motility. In particular, we found several proteins that have been linked to tumor progression and resistance, such as NPM. Baicalin treatment reduced the cellular proliferation rate and induced arrest during the S-phase of the cell cycle in A431 cells. NPM1 silencing significantly enhanced the effect of baicalin. Our data indicated that baicalin results in the significant inhibition of tumor growth in the A431 cell line, which may be associated with the regulation of the NPM gene expression.

[Effect of turmeric volatile oil on proliferation and apoptosis of human skin SCC A431 cells].

To investigate the effect of turmeric volatile oil (TVO) on the apoptosis and proliferation of human skin SCC A431 cells, A431 cells were incubated with different concentrations (5-80 mg•L⁻¹) of TVO in vitro．The proliferation and cell cycle were assessed by CCK8 assay. The change of morphology was observed with inverted microscope. Apoptosis was evaluated with AO/EB double staining and flow cytometry (FCM); cell cycle was analyzed with FCM ．Western blot method was used to detect caspase-3 and caspase-9 protein expression. Results indicated that TVO has significant inhibitory effects on the growth of A431 cells in a dose dependent relationship, the difference between groups has statistically significant (P<0.05). TVO group compared with control group, concentrations in cells shrivel and broken phenomenon, cell apoptosis rate increased, and a dose dependent and increased the expression of caspase-3 and caspase-9. The experiment results suggested that TVO could restrain skin squamous carcinoma A431 cells proliferation, and induce its apoptosis. The mechanism may be related to increase the expression of caspase-3 and caspase-9.

Effects of sirolimus and starved culture on autophagy of human A431 cells / 中华皮肤科杂志

Objective To evaluate effects of sirolimus(a classic autophagy inducer)and starved culture on autophagy of a human cutaneous squamous cell carcinoma cell line A431. Methods Cultured A431 cells and HeLa (a human cervical carcinoma cell line)cells were both classified into 5 groups to be treated with DMEM alone(control group), 0.1%dimethyl sulfoxide alone(DMSO group), 20 nmol/L sirolimus(20?nmol/L sirolimus group), 80 nmol/L sirolimus(80?nmol/L sirolimus group), and Earle′s balanced salt solution(EBSS group)respectively. After 4?hour treatment, Western blot analysis was performed to measure the expressions of autophagy?related markers microtubule?associated protein 1 light chain 3A/3B (LC3A/B) and recombinant gamma?aminobutyric acid receptor associated protein(GABARAP), and acridine orange staining to determine autophagy levels in these cells. Results As Western blot analysis showed, the ratio of LC3A/B?Ⅱto LC3A/B?Ⅰin A431 cells was similar between the control group and DMSO group(P > 0.05), but significantly higher in the 20?nmol/L sirolimus group, 80?nmol/L sirolimus group and EBSS group than in the control group(all P < 0.05). Western blot results from HeLa cells were similar to those from A431 cells. Bivariate correlation analysis revealed that the protein expression of GABARAP was positively correlated with that of LC3A/B ?Ⅰ in both HeLa cells(r = 0.869, 95% CI: 0.807 - 0.999, P = 0.051)and A431 cells(r = 0.837, 95% CI: -0.173 - 0.989, P = 0.037), but negatively correlated with that of LC3A/B?Ⅱ in both HeLa cells(r = -0.742, 95% CI: -0.982 - 0.406, P = 0.042)and A431 cells(r = - 0.684, 95% CI: -0.977 - 0.500, P = 0.047). Acridine orange staining showed that the percentages of autophagosome?positive A431 cells and HeLa cells were significantly increased in both the 80?nmol/L sirolimus group(23.750% ± 0.260% and 33.307% ± 0.715% respectively)and EBSS group(32.450% ± 0.488% and 66.097% ± 1.141% respectively) compared with the control group(15.987% ± 0.242% and 14.117% ± 0.295%, respectively, all P < 0.05). Conclusion The classic autophagy inducer sirolimus and starved culture can upregulate the autophagy level of A431 cells, and GABARAP may be highly correlated with LC3A/B.

Effects of inhibition of glucose-6-phosphate dehydrogenase expression on proliferation and cell cycle distribution of A431 cells / 中华皮肤科杂志

Objective To evaluate effects of downregulation of glucose?6?phosphate dehydrogenase(G6PD) expression on proliferation and cell cycle distribution of cutaneous squamous cell carcinoma(CSCC)cells. Methods Western blot analysis was performed to measure the protein expression of G6PD in normally cultured human HaCaT keratinocytes, SCL?1 and A431 CSCC cells. When A431 cells grew to 85%-90%confluence, a small interfering RNA (siRNA)targeting G6PD(G6PD?siRNA group)and a negative control siRNA(siRNA control group)were transfected into them separately, and untransfected A431 cells served as the untransfected group. CCK?8 assay was performed to evaluate proliferative activity of the A431 cells on days 0, 1, 2, 3 and 4 after transfection, Western blot analysis to measure G6PD, cyclin D1 and CDK4 protein expressions in A431 cells, and flow cytometry to analyze cell cycle distribution in A431 cells after 48 hours of additional culture. Results The protein expression of G6PD was significantly higher in normally cultured SCL?1 cells(0.308 ± 0.023)and A431 cells(0.643 ± 0.046)than in HaCaT cells(0.100 ± 0.019, both P 0.05). Compared with the untransfected group and siRNA control group, the G6PD?siRNA group showed significantly higher proportions of A431 cells in G0/G1 phase(both P < 0.001), but significantly lower proportions of A431 cells in S phase(both P<0.001). Conclusion G6PD may play important roles in the regulation of proliferation and cell cycle distribution of CSCC cells.