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Argemone mexicana(Pepaveraceae) is an important medicinal plant commonly known as 'maxican prickly poppy' and is traditionally used to treat skin diseases. In the present study, the extract/fractions of aerial parts of A. mexicana after carrying out the organoleptic characteristics were sequentially extracted with the solvents of increasing polarities. Total fractions were examined for their radical scavenging activities in DPPH and DNA nicking assays. Among all, maximum antioxidant activity was shown by chloroform fraction (AmC) in DPPH assay with IC50 of 26.12 μg/ml, and DNA nicking assay showed 80.91% protective potential. The AmC fraction was analyzed for its antibacterial, cytotoxic potential, cell cycle analysis, mitochondrial membrane potential (MMP) and accumulation of reactive oxygen species (ROS) using A431 cell line. The AmC fraction exhibited remarkable antibacterial activity against bacterial strains in the order Klebsiella pneumoniae> Bacillussubtilis> Salmonella typhi> Staphylococcus epidermidis. The cytotoxic potential of the AmC fraction was analyzed in skin epidermoid carcinoma (A431) cells, osteosarcoma (MG-63) and cervical (HeLa) cell lines with a GI50 value of 47.04 μg/ml, 91.46 μg/ml and 102.90 μg/ml, respectively. The AmC fraction was extended further to explore its role in cell death using A431 cell line. Phase contrast and scanning electron microscopic studies on A431 cells exhibited all the characteristics indicative of apoptosis, viz., viability loss, cell shrinkage, cell rounding-off, DNA fragmentation and formation of apoptotic bodies. Flow cytometric analysis revealed enhanced ROS level, decreased MMP and arrest cell cycle at the G0/G1 phase further strengthened cell death by apoptosis. Increased expressions of apoptotic markers (p53, PUMA, cyt c, Fas and Apaf-1) were confirmed by RT-qPCR analysis. Furthermore, the AmC fraction was subjected to ultra-high-performance liquid chromatography, which revealed the presence of different polyphenols in the order: caffeic acid> epicatechin> kaempferol> chlorogenic acid> gallic acid> catechin> ellagic acid >umbeliferone> quercetin> coumaric acid. A critical analysis of results revealed that the AmC fraction induced cell death in epidermoid carcinoma cells via ROS and p53-mediated apoptotic pathway which may be ascribed to the presence of polyphenols in it.

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.

Zinc oxide nanoparticles (ZnO-NPs) are used immensely in technology and medicine, but very less is known about toxicity mechanism to human epidermal cells. The objective of this study was to evaluate possible anticancer properties of ZnO-NPs on human epidermoid carcinoma cells using MTT assay, measurement of reactive oxygen species, DNA fragmentation, and nuclear condensation. ZnO-NPs were synthesized by sol-gel method using zinc acetate dihydrate, ethylene glycol, and 2-propyl alcohol. Numerous characterization techniques such as UV-visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, and dynamic light scattering spectroscopy were used to confirm synthesis, purity, optical, and surface characteristics, size, shape, and distribution of ZnO-NPs. Our finding showed that ZnO-NPs considerably decreased cell viability of human epidermoid carcinoma A431 cells with a parallel increase in nuclear condensation and DNA fragmentation in a dose dependent manner. Moreover, real time PCR expression study showed that treatment of human epidermoid carcinoma cells with ZnO-NPs trigger increased expression of tumor suppressor gene p53, bax, and caspase-3 while downregulate antiapoptotic gene bcl-2. Thus ZnO-NPs induce apoptosis in A431 cells through DNA degradation and generation of reactive oxygen species via p53, bax/bcl-2, and caspase pathways.

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

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

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.

Cancer cells can invade the surrounding stroma with the aid of fibroblasts (fibroblasts-dependent invasion). The aim of this study was to explore the spatiotemporal characteristics of fibroblast-dependent invasion of cancer cells. We performed an in vitro three-dimensional collagen invasion assay using Fluorescent Ubiquitination-based Cell Cycle indicator (Fucci)-labeled A431 carcinoma cells co-cultured with fibroblasts. We used time-lapse imaging to analyze the total cell number, frequencies of small cancer cell nests and S/G2/M phase of A431 cells in the invasion area. We compared the frequencies of small cancer cell nests and geminin (+) cancer cells within fibroblast-rich areas and fibroblast-poor areas in surgically resected human invasive squamous cell carcinoma tissue. The total invasion number of A431 cells was significantly higher when cultured with fibroblasts than without. The formation of small cancer cell nests was observed within the invasion area only in the presence of fibroblasts. The frequency of S/G2/M phase cells was significantly higher in A431 cells when cultured with fibroblasts than without. Immunohistochemical analysis of surgically resected human invasive squamous cell carcinoma tissue revealed that the frequencies of small cancer cell nests and geminin-positive cancer cells were significantly higher in fibroblast-rich areas compared to those in fibroblast-poor areas within the same tumor region. Our current study clearly showed that fibroblast-dependent cancer cell invasion was characterized by the progression in cell cycle and formation of small cancer cell nests.

Vitis vinifera. L is one of the most widely consumed fruits in the world and are rich in antioxidant abundant polyphenols. The present study was carried out to assess the antiproliferative and apoptotic effects of Vitis vinifera peel and seed extracts in an in vitro model using human epidermoid carcinoma A431 cell lines. Vitis vinifera peel and seed extracts were incubated with A431 cells to evaluate the antiproliferative, apoptotic effects and the morphological apoptotic changes induced by the extracts. Mitochondrial membrane potential was also measured after incubating the cells with extracts. At the inhibitory concentration (IC(50)), grape seed extract (111.11 µg/mL) and grape peel extract (319.14 µg/mL) were incubated for 24 h with A431 cells. Vitis vinifera peel and seed extracts were able to impart cytotoxic effects, induced apoptosis and apoptotic morphological changes in A431 cells significantly (p

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D3, i.e., 1α,25(OH)2D3 (1,25D3), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH)2D3, for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA-HO-1-n-1 and UE-HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D3. Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.

Since hedgehog (HH)/Gli1 that contributes to cancer proliferation and metastasis has been masked for decades, the signaling pathway was investigated about its exact role in proliferation and metastasis of cutaneous squamous cell carcinoma (SCC). Sonic hedgehog homolog (Shh), GLI family zinc finger 1 (Gli1), and vascular endothelial growth factor (VEGF) expressions in cutaneous SCC tissues were analyzed with immunohistochemistry, and their correlations with cutaneous SCC patients’ prognosis were conducted with Kaplan-Meier curve. Regarding in vitro studies, effects of the HH signaling pathway, and cyclopamine on patched 1 (Ptch1), smoothened/frizzled class receptor (Smo) and VEGF expressions were assessed in A431 cells based on western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Besides, Cell Counting Kit-8 (CCK-8) assay was implemented to evaluate cell proliferation, while wound-healing assay and transwell assay were performed to assess cell migration and invasion, respectively. Mice models were also established to observe effects of Gli1 on tumor diversity and incidence during a period of 20 weeks. Positively expressed VEGF, Gli1, and Shh proteins in cutaneous SCC tissues were correlated with poor survival of patients (P < 0.05). Besides, Gli1 messenger RNA (mRNA) and VEGF mRNA were observed to be significantly over-expressed in A431 cells (P < 0.05), and they were associated with incremental cell proliferation, invasiveness, and migration, which can be reversed by the interference of VEGF siRNA. Furthermore, cyclopamine treatment could induce inhibition of cell proliferation, invasiveness, and migration and suppression of Smo, Gli1, and VEGF expressions. The mice models also confirmed that Gli1 could significantly induce rise of tumor incidence and tumor diversity, while cyclopamine statistically relieved this transformation (P < 0.05). Abnormal activation of the HH signaling pathway plays critical roles in development of cutaneous SCC either in vivo or in vitro.

Skin cancer or cutaneous carcinoma, is a pre-eminent global public health problem with no signs of plateauing in its incidence. As the most common treatments for skin cancer, surgical resection inevitably damages a patient’s appearance, and chemotherapy has many side effects. Thus, the main aim of this study was to screen for a cell penetrating peptide (CPP) for the development of a targeting vector for skin cancer. In this study, we identified a CPP with the sequence NRPDSAQFWLHH from a phage displayed peptide library. This CPP targeted the human squamous carcinoma A431 cells through an interaction with the epidermal growth factor receptor (EGFr). Methyl-β-cyclodextrin (MβCD) and chlorpromazine hydrochloride (CPZ) inhibited the internalisation of the CPP into the A431 cells, suggesting the peptide entered the cells via clathrin-dependent endocytosis. The CPP displayed on hepatitis B virus-like nanoparticles (VLNPs) via the nanoglue successfully delivered the nanoparticles into A431 cells. The present study demonstrated that the novel CPP can serve as a ligand to target and deliver VLNPs into skin cancer cells.

Cutaneous squamous cell carcinoma (cSCC) is an epidermal keratinocyte-derived skin tumor, which is the second most common skin cancer in the general population. Recently, studies showed that microRNAs (miRNAs) played an important role in the development of cancer. In our study, we showed that the expression of SRCIN1 was lower in cSCC tissues than in the matched normal tissues. Moreover, there was significant inversed correlation between miR-346 and SRCIN1 in cSCC tissues. The luciferase reporter assay data showed that miR-346 can target the SRCIN1 message via the 3'-untranslated region (UTR) of SRCIN1. Overexpression of miR-346 inhibited the messenger RNA (mRNA) and protein expression of SRCIN1 in the A431 cells. In addition, ectopic expression of miR-346 promoted the A431 cell proliferation and migration. Meanwhile, SRCIN1 overexpression inhibited the A431 cell proliferation and migration. Rescue experiment has showed that SRCIN1 overexpression reduced the miR-346-induced A431 cell proliferation and migration. Herein, this study may provide miR-346 as a new therapeutic target for cSCC.

In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was “QAMXXMM-E/G/M-S/G-A/G/F-XG”. We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.

Inhibition of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACis) results in cancer cell growth inhibition, and HDACis have been revealed as potential anti-skin cancer agents. p21 is a cyclin-dependent kinase inhibitor and an essential regulator of growth inhibition. Recently, we reported that activating transcription factor 3 (ATF3) could significantly promote skin cancer cell growth. This study explored the relationship between ATF3 and HDACi-induced growth inhibition of epidermoid carcinoma cells. We found that trichostatin A (TSA) treatment inhibited cell growth in A431 epidermoid carcinoma cells in a dose-dependent manner. Simultaneously, p21 and ATF3 expression levels were upregulated and downregulated upon TSA stimulation, respectively. ATF3 overexpression promoted cell growth and downregulated p21 expression. In contrast, ATF3 depletion resulted in cell growth reduction and p21 transcriptional upregulation. More importantly, ATF3 overexpression partially antagonized TSA-induced growth inhibition and p21 activation. Collectively, these data demonstrate that ATF3 acts as an essential negative regulator of TSA-induced cell growth inhibition through interfering with TSA-induced p21 activation.

Cancer stem cells (CSCs) in most human tumors are commonly identified and enriched using similar strategies for identifying normal stem cells, including flow cytometry assays for side population, high aldehyde dehydrogenase (ALDH) activity, and CD133 positivity. Thus, development of a method for isolating a specific cancer using cancer-specific characteristic appears to be potentially important. Here, we reported extremely high Lgr5 levels in the specimen from skin squamous cell carcinoma (SCC) in patients. Using SCC cell line A431, we detected high Lgr5 and CD133 levels in ALDH-high or side population from these cancer cells. To figure out whether Lgr5 is a marker of CSCs in SCC, we transfected A431 cells with a Lgr5-creERT-2A-DTR/Cag-Loxp-GFP-STOP-Loxp-RFP plasmid and purified transfected cells (tA431) based on GFP by flow cytometry. 4-Hydroxytamoxifen (4-OHT) was given to label Lgr5-positive cells with RFP, for comparison to GFP-positive Lgr5-negative cells. Lgr5-positive cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Moreover, in Lgr5-negative population, Lgr5-positive re-appeared in culture with time, suggesting that Lgr5-positive cells can be regenerated from Lgr5-negative cells. Furthermore, the growth of tA431 cells significantly decreased upon a single dose of diphtheria toxin (DT)/4-OHT to eliminate Lgr5-positive cell lineage, while multiple doses of DT/4-OHT nearly completely inhibited tA431 cell growth. Taken together, our data provide compelling data to demonstrate that Lgr5-positive cells are CSCs in skin SCC.

MicroRNAs are a class of small non-coding RNAs that regulate the expressions of many genes. Previously, we found that the expression of p55PIK, an isoform of phosphatidylinosotol 3-kinase that has important roles in the regulation of cell cycle, is increased significantly in several types of cancer and contributes to the tumor growth. However, the mechanism for this increased p55PIK expression is not well understood. In this study, we show that miR-148b binds specifically to the 3'-untranslated region of p55PIK and significantly suppresses p55PIK expression. MiR-148b overexpression abolished p55PIK stimulation of cell proliferation and cell cycle progression in colorectal cancer (CRC) cell lines and decreased tumor growth in vivo. Furthermore, we demonstrated that p53 directly activates the transcription of miR-148b by binding to its promoter. In CRC cell lines and tissues, p53 expression was associated with miR-148b expression, and both were negatively associated with p55PIK expression. Our study shows that the p53/miR-148b/p55PIK axis has an important role in cell proliferation and tumor growth, and may represent a novel therapeutic target for treating cancers containing p53 mutations or losses.

Beta-urogastrone also known as human epidermal growth factor is a key member of epidermal growth factor family having role in cell proliferation and differentiation in vivo as well as in vitro. Human epidermal growth factor gene has been isolated from different tissues but the method of isolation is technically difficult and complicated as it deals with biopsies. Here we isolated mature partial human epidermal growth factor gene from Huh-7 cell line, amplified and abridged toward mature coding region with three steps PCR, sequenced for homology with wild type human epidermal growth factor gene, inbuilt with sites of interest and cloned in Pichia pastoris for expression study. Isolated mature human epidermal growth factor gene from Huh-7 cell line showed 100 % sequence homology to wild type human epidermal growth factor gene and gives the native expression for human epidermal growth factor peptide. In this study we report that Huh-7 cell line is an easy source for the particular gene of human epidermal growth factor isolation and we are also suggesting P. pastoris is an expression system to produce recombinant human epidermal growth factor of the therapeutic importance resembling to the natural human system.

The objective of this study was to determine the role and mechanism of S-phase kinase-associated protein 2 (Skp2) in colorectal cancer cell proliferation and survival both in vitro and in vivo. Adenoviral vector expressing Skp2 short hairpin RNA was transduced into SW480 cells. The effects of Skp2 on cell cycle and survival were assessed by Flow Cytometry. Cell proliferation was analyzed by MTT assay. The expression of cell cycle regulators p16 and p27 were measured by western blot. In vivo, human colorectal cancer was produced by xenograft of cancer cells in nude mouse. Tumor growth inhibitory rate was calculated to generate growth curve. Tumor growth was monitored by examining proliferating cell nuclear antigen expression, whereas tumor cell apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining. Knockdown of Skp2 blocked SW480 tumor cell growth and induced cell apoptosis. Skp2 appeared to be very important for the progression of cell cycle at G1/S phase. In vivo, blockade of Skp2 expression inhibited tumor growth and induced tumor apoptosis. Mechanistically, Skp2 regulated the expression of both p27 and p16 both in vitro and in vivo. The conclusion that we derive from this study is that Skp2 regulates colorectal cancer cell growth by inhibiting the expression of cell cycle regulator p27 and p16.

CKLF-like MARVEL transmembrane domain containing member 3 (CMTM3) is silenced in many kinds of cancers and inhibits tumor cells growth. We investigated the expression and role of CMTM3 in clear cell renal cell carcinoma (ccRCC). The expression of CMTM3 was detected in ccRCC tissue microarray, specimens, and cell lines by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. After transfected with CMTM3 plasmid or vector, the proliferation and migration of ccRCC 786-0 cells were determined by MTT assay and transwell assay, respectively. Furthermore, the anchorage-independent growth of transfected cells was assessed using soft agar colony formation assay. CMTM3 was down-regulated in 84 % (63/75) of ccRCC tissues and its expression had no correlation with the gender, age, clinical staging and histologic grade. CMTM3 protein was undetectable by western blot in most detected ccRCC specimens and two RCC cell lines (786-0 and ACHN). qRT-PCR analysis showed that CMTM3 mRNA was dramatically down-regulated in 40 ccRCC cancer tissues as compared with the paired adjacent normal ones. Restoration of CMTM3 significantly suppressed the anchorage-independent growth, proliferation and migration of 786-0 cells. These results indicate that CMTM3 is significantly down-regulated in ccRCC and exerts remarkable tumor-suppressive functions in 786-0 cells. Reduction of CMTM3 expression may contribute to the pathogenesis of ccRCC and CMTM3 may be a potentially target for therapeutic strategy.

In clinical studies, evaluating the combination of chemotherapy and the epidermal growth factor receptor (EGFR) inhibitor gefitinib, treatments were administered concurrently, despite it being counter-intuitive to give a cytostatic agent concurrent with cycle-active chemotherapy. One strategy to enhance efficacy might be to give the agents sequentially, thus allowing selective inhibition of repopulation of cancer cells between doses of chemotherapy. Here, we evaluate the hypothesis that sequential administration might allow inhibition of repopulation by gefitinib, with tumor cells re-entering cycle to allow sensitivity to subsequent chemotherapy. Sequential and concurrent administration of paclitaxel and gefitinib were studied in vitro and in xenografts using EGFR over-expressing, EGFR-mutant, and EGFR wild-type human cancer cell lines. We evaluated cell cycle distribution and repopulation during treatment. The sequential use of gefitinib and paclitaxel to treat EGFR over-expressing A431 cells in vitro decreased repopulation compared to chemotherapy alone, and there was greater cell kill compared to concurrent treatment. In contrast, combined treatment led to greater growth delay than use of gefitinib alone for concurrent but not for sequential treatment of mice bearing A431 xenografts; concurrent treatment had greater effects to reduce functional vasculature in the tumors. Conversely, sequential treatment led to greater growth delay than concurrent treatment of EGFR-mutant HCC-827 xenografts that are sensitive to lower doses of gefitinib. These studies highlight the importance of considering effects on the cell cycle, and on the solid tumor microenvironment, including tumor vasculature, when scheduling cytostatic and cytotoxic agents in combination.

In the present study, we report the effect and molecular mechanism of Ligularia fischeri (LF) on proliferation and migration in human lung cancer cells. LF-mediated inhibition of cell proliferation in p53 wild-type A549 and p53-deficient H1299 cells is accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G1 phase cell cycle arrest. In contrast, LF inhibits cell migration in A549 cells, but not in H1299 cells. These regulatory effects of LF on cell proliferation and migration are associated with inactivation of mitogenic signaling pathways such as ERK, Akt and p70S6K, and down-regulation of epidermal growth factor receptor and integrin β1 expression. Collectively, these findings suggest further development and evaluation of LF for the prevention and treatment of lung cancer with mutated p53 as well as wild-type p53.

Increasing evidence has demonstrated that the tumor suppressor gene deleted in liver cancer-1 (DLC1) is tightly implicated in the development and progression of tumors and is verified to be downregulated in a variety of tumors. However, the roles and precise molecular mechanisms of DLC1 in cutaneous squamous cell carcinoma (cutaneous SCC) remain to be elucidated. In the present study, we confirmed the reduced level in cutaneous SCC tissues and cells, and DLC1 mRNA relative level in cutaneous SCC tissues with lymph node metastasis (0.801 ± 0.079) was markedly lower than those without lymph node metastasis (1.245 ± 0.071) (P < 0.0001). Importantly, the survival rates of patients with low DLC1 level were lower than those with high DLC1 level (P = 0.0051). Further investigation revealed that DLC1 overexpression inhibited proliferation and arrested cell cycle at G0/G1 phase in A431 cells, which may be tightly associated with upregulation of p21 protein and downregulation of cyclin D1 and cdk2 proteins. Moreover, the decreases of FAK and p-FAK as well as the increase of E-cadherin level mediated by elevated DLC1 level suppressed invasion in A431 cells. Additionally, DLC1 overexpression induced apoptosis coupled with elevations of Bax level and caspase-3 activity and decrease of Bcl-2 level in A431 cells. Taken altogether, our data presented herein suggest that DLC1 plays a pivotal role in the development and progression of cutaneous SCC, which may be in part achieved by regulating the signaling pathway related to proliferation, invasion, cell cycle, and apoptosis in cutaneous SCC cells.

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

β-Ionone is an end ring analog of β-carotenoid which has been shown to possess potent anti-proliferative activity both in vitro and in vivo. To investigate the possible inhibitory effects of β-ionone, we studied cell growth characteristics, DNA synthesis, cell cycle progression, as well as mitogen-activated protein kinases (MAPKs) pathways in the human gastric adenocarcinoma cancer cell line (SGC-7901). Our results show that cell growth and DNA synthesis were inhibited, and the cell cycle was arrested at the G0/G1 phase in a dose-dependent manner in cells treated with β-ionone (25, 50, 100 and 200 μmol/L) for 24 h. We found that the β-ionone significantly decreased the extracellular signal-regulated kinase protein expression and significantly increased the levels of p38 and Jun-amino-terminal kinase protein expression (P < 0.01). β-Ionone also inhibited cell cycle-related proteins of Cdk4, Cyclin B1, D1 and increased p27 protein expression in SGC-7901 cells. These results suggested that the cell cycle arrest observed may be regulated through a MAPK pathway by transcriptional down-regulation of cell cycle proteins. These results demonstrate potent ability of β-ionone to arrest cell cycle of SGC-7901 cells and decrease proliferation.

In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.

Major events in cell biology are initiated by the binding of ligands to cell surface receptors and/or their transport into cells. We present a study of a simple microchannel system that integrates a bolus generator and surface-adhered cell culture domains. Our system allows the delivery of small packets or boluses of biomolecules to a cell population. Owing to pressure driven microfluidic flow of the bolus, a gradient of cell surface bound ligands is established along the length of the microchannel. Experimental data for the epidermal growth factor (EGF) binding to its receptor on A431 cells are presented. We highlight the effect of changing Peclet number (or flowrate), bolus shape, bolus volume and ligand concentration on the gradient formed longitudinally in the microchannel. A mathematical model describing the transient convection, diffusion, dispersion and binding of ligands to cell surface receptors is developed. The model provides essential design guidelines for our system with good qualitative agreement with experimental data. The results suggest ways to modulate the amount of bound ligand and the gradient independently. This simple microsystem is suitable for generating longer range gradients involving larger cell populations as compared to existing microfluidic systems.

Photodynamic therapy is widely used in the treatment of superficial skin cancers. 5-Aminolevulinic acid (ALA) and its methylated form, methyl-ALA (MAL), are frequently used as precursors to photosensitizing substances. Nevertheless, the mechanism of the uptake of ALA and MAL in keratinocytes and of their skin penetration is still controversial. Since both compounds are not sufficiently lipophilic to penetrate through lipid membranes, they must employ specific uptake systems which may vary between different cell types. Here, we studied ALA and MAL uptake in keratinocyte cell lines originating from healthy cells (CCD 1106 KERTr cells) or keratinocyte tumors (A431 cells). ALA uptake resulted in faster protoporphyrin IX (PpIX) production than MAL uptake. A pharmacological characterization of the uptake systems revealed that PpIX formation was most efficiently reduced with GABA transporter (GAT) substrates. GABA, β-alanine, and (S)-SNAP-5114 reduced ALA uptake and, to a lesser extent, MAL uptake in the cell lines. The pharmacology of these compounds indicates that ALA and MAL are taken up by normal and pathological keratinocytes via GAT-3. Furthermore, the amino acids arginine, cysteine, and histidine also inhibited the uptake of ALA, and even more so MAL, suggestive of an additional involvement of amino acid transporters. To show that PpIX formation in vivo is restricted to the application site, which has been questioned for ALA in one other report, we applied clinically used ALA and MAL formulations to the skin of nude mice. Contrary to the results of these previous authors, the resulting PpIX fluorescence increased over time and was restricted to the application site for both preparations.

Side population (SP) cells have been suggested to be multipotent cancer stem cells. To address whether SP cells exist in epidermal squamous cancer cell line A431, A431 cells dyed with Hoechst 33342 were sorted through flow cytometry. The SP cells were then analyzed by colony-forming and cell proliferation assay. Further, tumorigenicity and microarray analysis were used to compare biological difference between SP and non-SP (NSP) cells. Our results showed that SP cells existed in the A431 cell line, showing higher proliferating and colony-forming ability than NSP cells. Tumors generated from SP cells were larger than those from the NSP cells in NOD/SCID mice. The mRNA microarray profiling revealed that five cancer marker gene expressions were up-regulated and one tumor suppressor gene expression was down-regulated. These findings suggest that SP cells in A431 could contribute to self-renewal, neoplastic transformation, and cancer metastasis of human epidermal squamous cell carcinoma.

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Plant lectins have been reported to affect the proliferation of different human cancer cell line probably by binding to the specific carbohydrate moieties. In the present study, Badan labeled single cysteine mutant (present in the caveolin-1 binding motif) of jacalin (rJacalin) was found to penetrate the target membrane, indicating a protein-protein or protein-membrane interaction apart from its primary mode of binding i.e. protein-carbohydrate interaction. Further, Jacalin treatment has resulted in the movement of the GFP-Caveolin-1 predominantly at the cell-cell contact region with much restricted dynamics. Jacalin treatment has resulted in the perinuclear accumulation of PP2A and dissociation of the PHAP1/PP2A complex. PP2A was found to act as a negative regulator of ERK signaling and a significant decrease in the phosphorylation level of MEK and AKT (T308) in A431. In addition, we have also identified several ER resident proteins including molecular chaperones like ORP150, Hsp70, Grp78, BiP of A431 cells, which were bound to the Jacalin-sepharose column. Among various ER chaperones that were identified, ORP150 was found to present on the cell surface of A431 cells.

The understanding of the epidermal growth factor pathway in terms of intracellular signaling and its role in proliferation and cell survival has impacted the therapeutic management of many solid tumor malignancies in which this pathway has been dysregulated. Once the receptor is targeted at its cellular membrane level or tyrosine kinase domain, its blockage induces downregulation of oncogenic and tumorigenesis mechanisms which were in place, and thus inhibits proliferation and induces apoptosis of the malignant cell. Nowadays, we have several monoclonal antibodies as well as small molecules which target the receptor of epidermal growth factor. Although several receptors have been described within the human epidermal receptor family, our discussion will be focused on the impact of human epidermal receptor-1 as a therapeutic option for locally advanced squamous cell carcinoma of the head and neck.

To study the expression of VEGF, MMP-9, EGFR, and S100B in a highly brain metastases sub-clone cell line, PC14/B. The in vitro metastases-related behaviors of PC14 /B cells, such as adhesion to extracellular matrix (ECM), migration, and invasion were determined and compared with primary PC14 cells and A549 cells that do not metastasize to brain. The expression of vascular epithelial growth factor (VEGF), matrix metalloproteinase 9 (MMP-9), S100B, and epidermal growth factor receptor (EGFR) in the above three cell lines were measured by immunohistochemical staining and Western blot assay.The PC14/B cells have enhanced abilities of adhesion, migration, and invasion than PC14 cells and A549 cells. The expression levels of VEGF and MMP-9 in PC14/B cells are much higher than in PC14 and A549 cells. Two protein polymers of S100B are expressed specially in PC14/B cells. The expression of EGFR has a significant lower level in PC14 cells than in the other two cell lines. The increased expression of VEGF and MMP-9 may lead to the enhancement of adhesion, migration, and invasion of PC14/B cells. The expression of EGFR in PC14/B cells may have negative correlation with their capacities of metastasizing to brain. The specific expression of S100B in PC14/B cells strongly suggest that S100B might be a potential target for developing new therapy to brain metastases of lung cancer.

In this study, we characterized the effects of LA-12 on tumor cell lines possessing wild type p53 and on p53-deficient/mutant cell lines and the results were compared to those obtained using cisplatin. We have determined changes of p53 levels, of its transcriptional activity, of its posttranscriptional modifications and the effect of the treatment on the cell cycle, on the induction of apoptosis and on gene expression. LA-12 induces weak accumulation of both transcriptionally active p53 tumor suppressor and of p21(WAF1/CIP1) protein. LA-12 and cisplatin also significantly differ in their effects on apoptosis and cell cycle and on gene expression spectra in studied cell lines. LA-12 induces higher apoptosis levels in comparison with those induced by cisplatin, especially in p53-deficient H1299 cells and in MCF-7DD cells with transcriptionally inactive p53. We suggest that LA-12-mediated apoptosis is not fully dependent on p53. This confirms the therapeutic potential of LA-12 as a more potent cytostatic agent for both tumor cells expressing wild type p53 and for p53-deficient or mutant cells.

Activated epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target for a variety of solid tumors, particularly malignant gliomas. A recently discovered transmembrane glycoprotein, LRIG1, antagonizes the activity of epidermal growth factor receptor family receptor tyrosine kinases and acts as a negative feedback loop of EGFR and proposed tumor suppressors. The aim of this study was to investigate the impact of LRIG1 on the biological features of glioma cells and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1. We observed that the expression of LRIG1 was decreased, while the expression of EGFR was increased in the majority of astrocytomas, and the ratio of EGFR/LRIG1 was increased by sixfold in tumors versus corresponding non-neoplastic tissue. Upregulation of LRIG1, followed by a decrease of EGFR on the cytomembrane of the cells, induced cell apoptosis and cell growth inhibition, and further reversed invasion in glioma cell lines and primary glioma cells. Our study now clearly indicates that LRIG1 indeed affects cell fate and biology behaviors of the cells in vitro by inhibiting phosphorylation of downstream MAPK and AKT signaling pathway, and the elevated release level of caspase-8 might contribute to the enhanced apoptosis in LRIG1 transfected glioma cells. Taken together, these findings provide us with an insight into LRIG1 function, and we conclude that LRIG1 evolved in gliomas as a rare feedback negative attenuator of EGFR and could offer a novel therapeutic target to treat patients with malignant gliomas.

We present a mathematical model to study the effects of HER2 over-expression on cell proliferation in breast cancer. The model illustrates the proliferative behavior of cells as a function of HER2 and EGFR receptors numbers, and the growth factor EGF. This mathematical model comprises kinetic equations describing the cell surface binding of EGF growth factor to EGFR and HER2 receptors, coupled to a model for the dependence of cell proliferation rate on growth factor receptors binding. The simulation results from this model predict: (1) a growth advantage associated with excess HER2 receptors; (2) that HER2-over-expression is an insufficient parameter to predict the proliferation response of cancer cells to epidermal growth factors; and (3) the EGFR receptor expression level in HER2-over-expressing cells plays a key role in mediating the proliferation response to receptor-ligand signaling. This mathematical model also elucidates the interaction and roles of other model parameters in determining cell proliferation rate of HER2-over-expressing cells.

The effects of six new steroids of the pregnane series, with substituents at the 16α, 17α, and-or 3 and 19 positions of the steroid skeleton, on the viability of cultured breast cancer MCF-7 and cervical cancer HeLa cells were studied. The results showed that 3-keto-4-dehydrosteroids with a 16α,17α-spiropentane substituent and 3-keto-4-dehydro-16α,17α-cyclohexanopregnanes with 3-or 19-oxyimino groups decreased the viability of both types of cell culture. These compounds had negative influences on the expression of the epidermal growth factor receptor (EGFR) and induced apoptosis.

Epidermal growth factor (EGF) and transformation growth factor-alpha (TGFalpha) are potent mitogens that regulate proliferation of prostate cancer cells via autocrine and paracrine loops, and promote tumor metastasis. They exert their action through binding to the cell surface receptor, epidermal growth factor receptor (EGFR), and cause activation of Erk1/2 mediated mitogenic signaling in human prostate cancer (PCA) at both advanced and androgen-independent stages. Thus, we rationalized that inhibiting this mitogenic pathway could be useful in controlling advanced PCA growth.LNCaP and DU145 human PCA cells were treated with silibinin (100-200 microM) for different time points, and the levels of TGFalpha, activated signaling molecules (EGFR, Erk1/2 and Jnk1/2) and Erk1/2 kinase activity were analyzed employing ELISA, immunoprecipitation and/or immunoblotting techniques. The mRNA levels of TGFalpha were analyzed by RT-PCR.Treatment of cells (LNCaP and DU145) with silibinin resulted in a decrease in TGFalpha protein at both secreted and cellular levels together with a decrease in its mRNA level. Silibinin also caused an inhibition of EGFR activation followed by that of Erk1/2 without any change in their protein levels. The kinase activity of Erk1/2 to Elk1 was also inhibited by silibinin at least in DU145 cells. In other study, silibinin caused strong inhibition of Jnk1/2 activation in LNCaP cells while in DU145 cells, a strong induction in Jnk1/2 activation was observed. These results suggest that silibinin impairs TGFalpha-EGFR-Erk1/2 signaling in both androgen-dependent (LNCaP) and -independent (DU145) advanced human prostate carcinoma cells.This study, for the first time, identifies the inhibitory effect of silibinin on constitutively active TGFalpha-EGFR autocrine loop in advanced human PCA cells, which plausible contributes to the strong efficacy of silibinin in PCA prevention and intervention, as reported in recent studies.

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture supernatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture supernatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the G2/M phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As(2)O(3)) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (-84/-64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As(2)O(3)-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As(2)O(3 )treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As(2)O(3)-inhibited p21 expression, and then blocks the cell cycle arrest.

Growth factors and their receptors are significant to the biology of transitional cell carcinoma of the bladder. In this context, characterization of gene expression, amplification, and mutation has established the relevance of receptors as potential targets of novel therapeutic agents. Because these characteristics vary across disease states, they may serve as prognostic indicators of recurrence, progression, response to therapy, and mortality. This review focuses on the prognostic value of the human epidermal growth factor receptor family, including epidermal growth factor receptor (EGFR), human EGFR-2 (HER2), HER3, HER4, vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor-3 (FGFR3). EGFR and HER2 seem to indicate a poor prognosis, and HER4 and FGFR3 appear to be favorable prognostic indicators. However, validation studies are required to answer many remaining questions.

Despite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine. Cetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+®, a mesh nebulizer AeronebPro® and an ultrasonic nebulizer SYST’AM® LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells. The aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab. Altogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.

In this study, we present evidence that gene transfer of the CD40-ligand (CD154) into human immature dendritic cells (DC) imparts direct antitumor effects on tumor cells. DC infected with adenovirus directed to express human CD154 on the cell surface (CD154-DC) elicited significantly higher levels of immune accessory molecules commonly found on mature DC. We found that co-cultivation with a human squamous cell carcinoma cell line (OSC-70) with CD154-DC significantly inhibited cell growth. We further demonstrate that OSC-70 cells stimulated with CD154-DC were more susceptible to apoptosis via TNF-related apoptosis inducing ligand (TRAIL). Importantly, tumor cells co-cultured with CD154-DC in transwell plates expressed upregulated cell surface TRAIL-R2. CD154-DC produced higher levels of interferon (IFN)-gamma, IL-12p70 and soluble CD154, but the ability of CD154-DC to inhibit tumor cell growth was significantly abrogated by a neutralizing antibody to IFN-gamma, indicating that this was mainly mediated by IFN-gamma. Furthermore, intratumoral injection of CD154-DC significantly suppressed OSC-70 tumor growth in a xenograft model. Overall, these results reveal that CD154-DC have potential as an anti-cancer therapy by producing IFN-gamma to arrest adjacent tumor cell growth and increase the susceptibility of apoptosis via TRAIL.

Pdcd4 (programmed cell death protein 4) was identified as a gene up-regulated during apoptosis and, additionally, seems to have a function as a tumor suppressor. However, there are conflicting data concerning its role in programmed cell death and most results for its action as an inhibitor for neoplastic transformation are derived from experiments with epidermal cells. Therefore, we were interested to investigate if the action of Pdcd4 might be cell type specific. For that purpose we examined the expression of Pdcd4 and several other proteins in various tumor cell lines. We could not find any correlation of Pdcd4 levels and expression of proteins associated with cell cycle and/or apoptosis in different cell lines. Furthermore, we stably transfected two cell lines (Bon-1 and HCT116) to over-express Pdcd4 and analyzed protein expression. Although we found several regulated proteins none of these proteins were affected in both cell lines in the same manner. For instance, dUTPase expression was reduced in Bon-1 cells but not changed in HCT116 cells. This regulation might be important for the sensitivity of cells to anti-cancer drugs like inhibitors of thymidilate synthase. Therefore, we conclude that the function of Pdcd4 might be cell type specific. A role for Pdcd4 in apoptosis or as a tumor suppressor might be limited to certain cell types.

The aim of this study was to investigate the role of pirh2 (p53-induced RING-H2) protein in the proliferation, apoptosis and cell cycle control of the lung cancer cell line A549. Pirh2 expression was detected by immunofluorescence, Western blot analysis and real-time polymerase chain reaction (PCR). Cell proliferation was assessed by cell counting kit-8 (CCK-8). Cell cycle control and apoptosis were analyzed by flow cytometry. The results showed that pirh2 was expressed in the cytoplasm of A549 cells. The inhibition of pirh2 expression by siRNA (psiRNA-pirh2) resulted in reduced cell proliferation and increased apoptosis. In addition, the number of G0/G1 phase cells was increased but G2/M cells were not affected significantly. Taken together, the inhibition of pirh2 expression in the lung adenocarcinoma cell line A549 resulted in reduced tumor cell growth via the inhibition of cell proliferation, the activation of apoptosis and the interruption of cell cycle transition.

Several human tumors such as neuroendocrine tumors, medullary thyroid carcinoma, etc., express somatostatin receptors which specifically bind somatostatin and its analogues such as lanreotide, octreotide, etc. In order to prepare a therapeutic agent for targeting such tumors, attempts were made to prepare 90Y-DOTA-Lanreotide. Lanreotide could be successfully conjugated with the macrocyclic chelating agent DOTA (1,4,7,10-tetraaza cyclododecane tetracetic acid) which forms stable complexes with 90Y. 90Y-DOTA-Lanreotide could be prepared in >98% radiochemical purity and remained stable for 72 hours at room temperature. The tracer showed specific binding to A431 cells. Biodistribution studies in C57BL6 mice bearing melanoma showed ∼1.3% uptake pergram of tumor at 24-hour p.i.

We have previously reported that As(2)O(3) affected cell cycle progression and cyclins D1 and B1 expression in two glioma cell lines differing in p53 status (U87MG-wt; T98G-mutated). In the present study, we further demonstrated that As(2)O(3) affected proliferation, viability and apoptosis of the two cell lines in a dose- and time-dependent manner, and T98G cells were more sensitive than U87MG cells to As(2)O(3) -induced apoptosis and inhibition of proliferation and viability. We further investigated the expression profiles of genes related with apoptosis and cell cycle in the two cell lines with a human cDNA-microarray (SuperArray) spotted with 267 genes of apoptosis and cell cycle. Thirty five genes were upregulated and 15 genes downregulated at least 2-fold by As(2)O(3) in U87-MG cells; whereas, 38 genes were upregulated and 21 genes downregulated at least 2-fold in T98G cells by As(2)O(3). After As(2)O(3) treatment, p53 expression was upregulated 56.5-fold in T98G cells, but only 6.0-fold in U87MG cells. The results indicate that As(2)O(3) suppresses the growth of U87MG cells mainly by regulating expression of genes of cell cycle arrest, stress and toxicity; whereas As(2)O(3) affects T98G cells mainly by regulating expression of genes belonging to Bcl-2, tumor necrotic factor receptor and ligand families. The data may be helpful for optimizing As(2)O(3) as an anti-cancer drug in the treatment of gliomas.

Recently, we demonstrated that S100C/A11 comprises an essential pathway for growth suppression by TGFbeta in normal human keratinocytes. Nuclear transfer of S100C/A11 was a hallmark of the activation of the process. In the present study, we examined the possible deterioration in the pathway in human squamous cancer cell lines, focusing on intracellular localization of S100C/A11 and its functional partners Smad3 and Smad4. All four human squamous cancer cell lines examined (A431, BSCC-93, DJM-1, and HSC-5) were resistant to growth suppression by TGFbeta. In BSCC-93, DJM-1, and HSC-5 cells exposed to TGFbeta, S100C/A11 was not transferred to the nuclei, and p21(WAF1) was not induced. Overexpression of nucleus-targeted S100C/A11 partially recovered induction of p21(WAF1) and p15(INK4B) and growth suppression by TGFbeta1 in these cells. These results indicate that the deterioration in the S100C/A11-mediated pathway conferred upon the cancer cell lines resistance to TGFbeta. In A431 cells, S100C/A11, Smad3, and Smad4 were simultaneously transferred to the nuclei, and p21(WAF1) was induced upon exposure to TGFbeta. We provide evidence to indicate that refractoriness of A431 cells to TGFbeta was probably because the amount of p21(WAF1) induced by TGFbeta was insufficient to counteract cyclin A, which is highly overexpressed in A431 cells. Thus, the newly found S100C/A11-mediated pathway is at least partly involved in conferring upon human squamous cell cancers resistant to TGFbeta-induced growth suppression, which is considered to play a critical role for the initiation and progression of many human cancers.

The epidermal growth factor receptor (EGFR) autocrine pathway plays a crucial role in human cancer, contributing to a number of highly relevant processes in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis, and metastatic spread. For these reasons, the EGFR is one of the most studied and exploited targets for molecular cancer therapy. Two classes of anti-EGFR agents have entered clinical practice: monoclonal antibodies and small molecules targeting the receptor tyrosine kinase. They have shown activity alone and in combination with conventional antitumor treatments, such as chemotherapy or radiation therapy. However, preclinical and clinical studies have demonstrated the occurrence of spontaneous or acquired resistance to these drugs. Since EGFR is implicated in a wide array of intracellular functions, the phenomenon of resistance to its inhibitors may result from the derangement of different molecular pathways, including autocrine/paracrine production of ligands, receptor mutations, constitutive activation of downstream signaling proteins, and activation of alternative pathways. This review presents the experimental evidence underlying the main mechanisms of resistance to EGFR inhibitors.

Nicotinic acid (niacin) has been used clinically to manage dyslipidemia for many years. The molecular target of nicotinic acid was unknown until the recent revelation of human G-coupled receptor HM74a as the high affinity receptor for nicotinic acid. In searching for a cell line expressing endogenous human HM74a receptor, we have identified that the A431 cell line, a human epidermoid cell line, expresses a high level of HM74a receptor. An HM74a-specific real time PCR probe set was designed and the mRNA levels of HM74a in A431 and 32 other cultured cell lines were measured quantitatively. When the mRNA expression of HM74a in A431 cells was compared to that in human primary preadipocytes, adipocytes and adipose tissue, we found that the level in A431 was about 10- fold higher than that in adipocytes and adipose tissue. The ratio of HM74a:HM74 mRNA was measured quantitatively and it was determined to be 3:2 in A431 cells. The function of the HM74a receptor in A431 cells was evaluated for its ability to inhibit forskolin-induced cAMP production. Pertussis toxin treatment abolished the inhibition. Our data suggest that the A431 cell line may serve as a cellular model for further investigation of niacin/HM74a-mediated signal transduction in modulating metabolism. A431 cell line may also provide a valuable cell model to study prostaglandin production upon HM74a activation to improve our understanding of niacin/HM74a-mediated skin flushing.