Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.     

RNA干扰DNA修复提高肿瘤细胞的放疗敏感性

放射性抵抗是目前肿瘤放射治疗中难以解决的一大难点。放射损伤的作用靶点为DNA, DNA损伤后,会启动自身的修复系统进行修复,能针对自身不同类型的DNA损伤启动不同修复机制,DNA的修复在一定程度上导致了放疗抵抗的发生。近年来,RNA干扰在肿瘤、病毒性疾病和遗传病等基因治疗研究方面均取得了一定的成果,早在1968年,Alexander就提出了细胞辐射敏感性取决于其DNA链断裂修复能力的概念。相关研究证明,通过构建表达dsRNA或者siRNA来干扰DNA修复蛋白,如Ku二聚体(Ku70和Ku80)、DNA-PKcs、Ku、ATM、Rad51、BRCA1、P53、XRCC4等的表达,联合放疗,都在不同程度上增加了放疗的敏感性。本文就RNA干扰DNA修复促进肿瘤细胞放疗敏感性的研究进展作一综述,并对其应用于肿瘤临床治疗的前景提出展望。     

Effect of epidermal growth factor on clonogenic growth of primary human tumor cells

The effect of EGF on the soft agar colony-forming ability of fresh human tumor cells was assessed in 40 specimens obtained from various types of carcinoma including those of the breast, endometrium ovary, and other sites. Cells from four established human tumor cell lines (three breast and one endometrial) were also included in this study. The results showed that addition of EGF at a concentration of 50 ng/ml resulted in a 50% higher cloning efficiency in soft agar in 40% of the samples of fresh human tumors. When cells from tumor cell lines were plated in semi-solid medium containing EGF, the number of colonies formed was at least twice as high as controls. Cells from fresh tumor biopsies were assayed for EGF receptors to determine whether the correlation between the proliferative response in EGF-supplemented semi-solid medium as compared to control could be related to the number of EGF receptors present on the cells. Specific receptors for EGF were detected by using radioiodinated EGF in early-passage cell cultures from some of the tumors tested for clonogenicity. The number of receptors ranged from 0.3 to 3.27 X 10(5) per cell. Cells from two melanoma specimens possessed less than 0.3 X 10(3) EGF receptors per cell. We found no correlation between the number of EGF receptor on a cell surface and the mitogenic effect of EGF on the same tumor cells grown in semi-solid medium.     

Blockage of epidermal growth factor receptor by quinazoline tyrosine kinase inhibitors suppresses growth of human hepatocellular carcinoma

Epidermal growth factor receptor (EGFR) is highly expressed in many human tumors including hepatocellular carcinoma (HCC). Therefore, inhibition of EGF receptors could be a potential target for anticancer therapy. In this study, we investigated the effects of two EGFR tyrosine kinase inhibitors, PD153035 and its analogue 4-[[3-chloro-4-fluorophenyl]amino]-6,7-dimethoxyquinazoline hydrochloride (ANAPD) on human HCC cell lines by cell proliferation assay, flow cytometry and Western blot. Our results demonstrated that both EGFR inhibitors inhibited tumor cell growth in a dose-dependent manner, but ANAPD was more potent than PD153035. These specific inhibitors not only blocked EGF-stimulated EGFR autophosphorylation but also targeted EGFR signaling including MAPK and Akt pathways. Furthermore, EGFR inhibitors induced a delay in cell cycle progression and a G(1) arrest together with a partial G(2)/M block. EGFR inhibitors also induced tumor cells to undergo apoptosis. In conclusion, this study demonstrated that both PD153035 and ANAPD inhibit tumor cell growth in HCC through inhibition of EGFR signaling pathway, and ANAPD is a more potent inhibitor than PD153035. This suggested that blockage of EGF receptors may provide an effective therapeutic approach for human HCC and ANAPD could be a potential drug candidate for the treatment of HCC.     

Targeting fibroblast growth factor receptor 3 enhances radiosensitivity in human squamous cancer cells

Conventional therapies including radiation therapy cannot cure squamous cell carcinoma (SCC), and new treatments are clearly required. Our recent studies have shown that SCC cell lines exhibiting radioresistance show significant upregulation of the fibroblast growth factor receptor 3 (FGFR3) gene. We hypothesized that inhibiting FGFR3 would suppress tumor cell radioresistance and provide a new treatment approach for human SCCs. In the present study, we found that RNA interference-mediated FGFR3 depletion in HSC-2 cells, a radioresistant cell line, induced radiosensitivity and inhibited tumor growth. Use of an FGFR3 inhibitor (PD173074) obtained similar results with suppression of the autophosphorylation extracellular signal-regulated kinase pathway in HSC-2 cells and lung cancer cell lines. Moreover, the antitumor growth effect of the combination of PD173074 and radiation in vivo was also greater than that with either drug alone or radiation alone. Our results provided novel information on which to base further mechanistic study of radiosensitization by inhibiting FGFR3 in human SCC cells and for developing strategies to improve outcomes with concurrent radiotherapy.     

microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling

Many microRNAs (miRNAs) play crucial roles in regulating expression of oncogenes or tumor suppressor genes. The epidermal growth factor receptor (EGFR) is frequently overexpressed in a wide range of solid tumors and is an important therapeutic target; however, the therapeutic outcome of currently available anti-EGFR agents is often limited due to diverse molecular resistance mechanisms. In this study, we evaluated the potential of targeting miRNA-7 for overcoming radio-resistance of cancer cells with activated EGFR-associated signaling. A panel of human cancer cell lines with increased EGFR-PI3K-Akt signaling was transfected with pre-miR-7 or control miRNA. Ectopic overexpression of miR-7 attenuated EGFR and Akt expression and radiosensitized SQ20B squamous cell carcinoma of the larynx, MDA-MB-468 breast cancer cells, A549 lung carcinoma cells, and U251 and U87 malignant glioma cells. In contrast, antisense-mediated inhibition of mature miR-7 expression led up-regulation of EGFR and its downstream effectors, and increased radio-resistance of U251 glioma cells. Overexpression of miR-7 prolonged radiation-induced γH2AX foci formation and downregulation of DNA-dependent protein kinases (DNA-PKcs). miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers with activated EGFR-PI3K-AKT signaling.     

Characterization of human epidermal growth factor receptor and c-Src interactions in human breast tumor cells

In C3H/10T1/2 murine fibroblasts, overexpression of both c-Src and the human epidermal growth factor (EGF) receptor 1 (HER1) is required for detection of stable complexes between the two molecules and results in hyperactivation of the receptor and synergistic increases in tumor formation in nude mice, as compared with cells that overexpress only one of the pair. Elevated levels or activities of c-Src and HER1 also occur in a subset of later-stage breast cancers, suggesting that interactions between these two molecules could contribute to a more aggressive clinical course. To determine whether stable complexes between c-Src and HER1 occur in human breast cancers under the same conditions as in murine fibroblasts and whether the appearance of such complexes correlates with enhanced signaling through the EGF receptor and increased tumor growth, human breast tumor cell lines and tumor tissues were analyzed for a number of c-Src/HER1–mediated signaling events and tumorigenicity. In a panel of 14 cell lines, 10 overexpressed c-Src, and of these, five contained elevated levels of HER1 and exhibited an EGF-dependent association between HER1 and c-Src. This association was also present in a HER1/c-Src–overexpressing tumor sample from a breast cancer patient. Further analysis of signaling events revealed that phosphorylation of the HER1 substrate, Shc, and its downstream effector, mitogen-activated protein kinase, was increased in EGF-stimulated MDA-MB-468, MDA-MB-231, and BT-549 cells (which overexpress both c-Src and HER1) as compared with MCF7 and ZR-75-1 cells (which only overexpress c-Src). Furthermore, MDA-MB-468 and MDA-MB-231 cells displayed increased tumorigenicity in nude mice. These results support the hypothesis that c-Src/HER1 interactions contribute to tumor progression in certain late-stage breast tumor cells. Mol. Carcinog. 21:261–272, 1998. © 1998 Wiley-Liss, Inc.     

Shikonin modulates cell proliferation by inhibiting epidermal growth factor receptor signaling in human epidermoid carcinoma cells

Shikonin isolated from the roots of the Chinese herb Lithospermum erythrorhizon has been associated with anti-inflammatory properties. We evaluated shikonin's chemotherapeutic potential and investigated its possible mechanism of action in a human cutaneous neoplasm in tissue culture. Shikonin preferentially inhibits the growth of human epidermoid carcinoma cells concentration- and time-dependently compared to SV-40 transfected keratinocytes, demonstrating its anti-proliferative effects against this cancer cell line. Additionally, shikonin decreased phosphorylated levels of EGFR, ERK1/2 and protein tyrosine kinases, while increasing phosphorylated JNK1/2 levels. Overall, shikonin treatment was associated with increased intracellular levels of phosphorylated apoptosis-related proteins, and decreased levels of proteins associated with proliferation in human epidermoid carcinoma cells.     

Inhibition of epidermal growth factor binding to cultured mouse epidermal cells by tumor promoters

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) caused partial inhibition of the binding of [125I]epidermal growth factor ([125I]EGF) to intact mouse epidermal cells (HEL/37). The proportion of [125I]EGF binding which was insensitive to TPA inhibition decreased with increasing EGF concentration. The partial inhibition of [125I]EGF binding was not due to destruction of TPA or to covalent linkage of EGF to receptor sites. The binding of [125I]EGF to HEL/37 cells showed a curvilinear Scatchard plot; this was converted into a linear plot in the presence of TPA. We conclude that TPA acts to inhibit interactions between EGF receptors or between EGF receptors and some other membrane component(s).     

Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.     

Inhibition of phosphatidylinositol 3-kinase signaling negates the growth advantage imparted by a mutant epidermal growth factor receptor on human glioblastoma cells

In de novo glioblastoma multiforme, loss of the tumour suppressor protein PTEN can coincide with the expression of a naturally occurring mutant epidermal growth factor receptor known as deltaEGFR. DeltaEGFR signals constitutively via the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt and mitogen-activated protein kinase pathways. In human U87MG glioblastoma cells that lack PTEN, deltaEGFR expression enhances tumourigenicity by increasing cellular proliferation. Inhibition of PI3K signaling with the pharmacologic inhibitor wortmannin, or by the reconstitution of physiological levels of PTEN to dephosphorylate the lipid products of PI3K, negated the growth advantage imparted by deltaEGFR on U87MG cells. PTEN reconstitution suppressed the elevated PI3K signaling, without affecting mitogen-activated protein kinase signaling and caused a delay in G1 cell cycle progression that was concomitant with increased cyclin-dependent protein kinase inhibitor p21CIP1/WAF1 protein levels. Our study provides insight into the mechanism by which deltaEGFR may contribute to glioblastoma development.     

Targeting phthalocyanines to tumor cells using epidermal growth factor conjugates.

Epidermal growth factor (EGF) was used as a vector for targeted delivery of phthalocyanines to tumor cells. The conjugates of EGF with disulfochloride aluminum phthalocyanine [Pc(Al)] and disulfochloride cobalt phthalocyanine [Pc(Co)] were synthesized. The cytotoxic activity of the conjugates against the human breast carcinoma cell line MCF-7 was determined. The cytotoxic activity of the EGF-Pc(Co) conjugate was 4.5 times higher than that of the EGF-Pc(Al) conjugate. The antitumor activity of the EGF-Pc(Co) conjugate was also studied in vivo in murine melanoma B16. Compared to free Pc(Co), intravenous injections of Pc(Co) conjugated with EGF inhibited tumor development and increased mean life span and mean survival time of experimental animals.     

BSO-induced reduction of glutathione levels increases the cellular radiosensitivity of drug-resistant human tumor cells.

Acquired resistance to cis-platinum and melphalan, in the human ovarian OAW42 tumor cell line, respectively, conferred a 3- and 1.5-fold decrease in photon sensitivity. Analysis of cell survival curves by the linear quadratic equation showed an accompanying 5- and 2-fold reduction in the magnitude of the initial slope (alpha). Treatment with the GSH depleting agent BSO restored the magnitude of alpha to a value similar to that of the parental line without evidence of dose modification in the high-dose region of the cell survival curve. This in conjunction with failure of alteration in GSH levels to affect parental OAW2 sensitivity and of the SER of BSO to reflect GSH levels suggest a possible GSH independent mechanism of action for BSO. If similar patterns occur in the clinic, the possibility exists of circumventing collateral resistance between chemotherapeutic agents and ionizing radiation, provided that tumor thiol levels can be preferentially depleted.     

Antisense epidermal growth factor receptor delivered by adenoviral vector blocks tumor growth in human gastric cancer.

Epidermal growth factor receptor (EGFR) protein overexpression is commonly found in human gastric cancer, and its gene amplification is known to correlate with poor prognosis in gastric cancer patients. With regard to therapy trials targeting EGFR, it has been reported that stable transfection of EGFR antisense or treatment with antibody against EGFR results in growth suppression of human cancer cells that express high levels of EGFR. We have designed an adenovirus-expressing antisense EGFR and have investigated its effect on the growth of gastric cancer in vitro and in vivo. Following infection with EGFR antisense RNA-expressing adenovirus (Ad-EAS), the cell surface EGFR protein levels of infected cancer cells were markedly reduced, and the in vitro growth of Ad-EAS-infected cells was significantly inhibited relative to control-infected cells in all three gastric cancer cell lines (AGS, KKLS, and MKN28) studied here (P < .0002). In a nude mouse subcutaneous tumor system, in vivo tumor growth of MKN28 was significantly inhibited after Ad-EAS treatment, and inhibition on day 48 was 93% by volume compared with that of untreated controls. These results suggest that an adenoviral vector system targeting the down-regulation of EGFR could be a good candidate for the therapy of gastric cancers that overexpress EGFR.     

Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells

Overexpressed epidermal growth receptor factor receptors (EGFRs) are thought to contribute to the malignant phenotype of human glioblastomas (GBMs), but the mechanism is not well understood. We found that SKMG-3 cells, a rare GBM cell line that maintains EGFR gene amplification in vitro, produced high levels of EGFR protein. The cells also expressed the related receptors HER2/neu and HER4, but not HER3. Immunoblots and tryptic phosphopeptide maps showed that the SKMG-3 EGFRs were intact and functional and that a subset of these receptors were spontaneously autophosphorylated. EGF treatment stimulated phosphorylation of the EGFRs as well as the downstream effectors Erk, AKT1, stat3 and c-Cbl. Under minimal growth conditions, the unstimulated SKMG-3 cells contained constitutively phosphorylated Erk and AKTI but no detectable stat3 DNA-binding complexes. The EGFR kinase inhibitor PD158780 reduced the constitutive phosphorylation of the receptor and Erk but not that of AKT1. In contrast, inhibition of phosphatidylinositol-3-kinase (PI3K) blocked the constitutive phosphorylation of Erk and AKT-1 but not the EGFR. We conclude that the SKMG-3 cells represent the subset of GBMs with amplified EGFR genes that overexpress intact receptors. The results also suggest that in some GBMs, signals from overexpressed EGFRs contribute to the constitutive phosphorylation of Erk, but these signals may not required for the constitutive activation of PI3K or AKT1.     

SLUG silencing increases radiosensitivity of melanoma cells in vitro

Background

Melanoma radioresistance has been attributed to the presence of tumor cells with highly efficient DNA damage repair mechanisms. We examined the expression of genes involved in DNA damage repair and DNA damage sensing, and assessed their modulation by SLUG silencing, which is potentially capable of increasing radiosensitivity.

Methods

Two melanoma cell lines (M14 and M79) were used to evaluate in vitro radiation-induced cytotoxicity before and after SLUG silencing. mRNA expression levels of BRCA1, ERCC1, DNA-PK, PARP, MGMT, ATM and TGM2 were determined by real-time RT-PCR, and protein expression levels of SLUG, caspase 3, p21, PUMA and pMAPK by Western blotting.

Results

The cytotoxic effect of radiation was high in M14 and low in M79 cells. SLUG silencing increased the interference of radiation on cell cycle distribution and cell killing by 60 % and 80 % in M79 cells after a 2.4 Gy and 5 Gy radiation dose, respectively. It also led to a significant inhibition of expression of genes involved in DNA damage repair and DNA damage sensing in all cell lines maintained after radiation. An almost total inhibition was observed for TGM2, which is expressed at a high basal level in the most radioresistant cell line (M79). Protein expression of PUMA was induced by radiation and was enhanced after SLUG silencing.

Conclusions

Our results reveal a pivotal role of SLUG in regulating a cellular network involved in the response to DNA damage, and highlight the importance of TGM2 in radiosensitivity modulation. SLUG silencing appears to increase radiation sensitivity of the melanoma cells tested.     

Resistance of human cervical carcinoma cells to tumor necrosis factor correlates with their increased sensitivity to cisplatin: evidence of a role for DNA repair and epidermal growth factor receptor.

Alterations in cellular biochemistry which are associated with the development of resistance to cytotoxic peptides, such as tumor necrosis factor (TNF), may also be responsible for changes in the response of cells to cytotoxic agents. Culturing ME-180 cervical carcinoma cells in the presence of escalating concentrations of TNF resulted in the development of an ME-180 cell variant (ME-180R) resistant to TNF but expressing a 3-5-fold increased sensitivity to cisplatin (CDDP) when measured following continuous exposure (low doses) or short-term incubation with CDDP (high doses) and clonogenic analysis. Cellular platinum uptake, efflux, and nuclear platinum content as well as the extent of DNA platination were examined and found to be identical in both ME-180 parental and ME-180R cell lines. Although ME-180R cells showed a relatively higher glutathione content than ME-180 parental cells, the effect of buthionine sulfoximine on the cellular sensitivity to CDDP and glutathione S-transferase activities of both cell lines were almost identical, suggesting that glutathione content or its metabolism did not appear to play a major role in differential CDDP cytotoxicity. Unscheduled DNA synthesis following exposure to CDDP was more inducible in ME-180 parental cells than in CDDP-sensitive ME-180R cells. Alkaline elution studies of cross-linked DNA in CDDP-treated ME-180 cells suggested that accumulation of DNA adducts reached maximal levels 10-15 h after CDDP treatment and was similar in both TNF-resistant and parental cells. Within 24 h after CDDP exposure, the extent of DNA cross-linking was markedly reduced in parental cells but remained elevated in the CDDP-sensitive ME-180R cell line. To examine the proposed regulatory role of phosphorylation in CDDP and TNF-mediated cytotoxicity, epidermal growth factor (EGF) receptor tyrosine kinase activity was measured in both TNF-resistant and parental ME-180 cells. Analysis of cell lysates demonstrated a 3-4-fold higher EGF receptor tyrosine kinase activity in ME-180R cells when compared to the parental population which correlated with increased expression of EGF receptor protein by immunoblot analysis. Based upon colony-forming assays, EGF treatment of ME-180 parental cells resulted in an increased sensitivity to CDDP (similar to ME-180R cells) and 3-fold stimulation of EGF receptor tyrosine kinase activity. Taken together, these results suggest that TNF resistance in ME-180 cervical carcinoma cells correlates with both increased EGF receptor expression and enhanced CDDP cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)