Expression of apoptosis-related genes in human head and neck squamous cell carcinomas undergoing p53-mediated programmed cell death.

Human head and neck squamous cell carcinoma (HNSCC) lines infected with a replication-defective Ad5CMV-p53 vector bearing a wild-type human p53 gene were used to examine alterations in the production of proteins implicated in regulating apoptosis. Because HNSCC lines express abundant levels of c-myc, and simultaneous expression of c-myc and p53 is known to trigger apoptosis in other cells, cooperation between these two genes was examined. Surprisingly, levels of c-myc mRNA and protein were rapidly and profoundly suppressed after infection with wild-type p53. Suppression of c-myc using antisense oligodeoxynucleotides (in the absence of p53) was sufficient to trigger apoptosis in Tu-138 cells, raising the possibility that the reduction of c-myc may be involved in at least one of the cell death pathways mediated by p53. Expression of a panel of Bcl-2 homology proteins was also examined in HNSCC lines undergoing p53-mediated apoptosis. No changes in Bcl-2, Bak, or Bcl-xS were found after p53 expression. Increased levels of the apoptosis-accelerating protein Bax were found in HNSCC lines after infection with Ad5CMV-p53. Induction of the apoptosis-inhibiting protein Bcl-xL was observed in Tu-167 cells and may account for the delayed onset of apoptosis in these cells. These studies suggest that multiple pathways may regulate apoptosis after transient overexpression of p53.     

Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck

We examined effects of the anti-epidermal growth factor receptor monoclonal antibody C225 on proliferation, cell cycle phase distribution, apoptosis, and radiosensitivity in squamous cell carcinoma (SCC) cell lines derived from head and neck cancer patients. Exposure to C225 in culture inhibits SCC proliferation in a time-dependent manner, and the degree of growth inhibition, compared to controls, ranges from 20 to 75%. Flow cytometry analysis demonstrates that C225 treatment induces accumulation of cells in G1, which is accompanied by a 2-3-fold decrease in the percentage of cells in S phase. C225 exposure also induces apoptosis in SCC populations, as demonstrated by flow cytometry analysis using dual stainings of merocyanine 540 and Hoechst 33342. Western blot analysis indicates that C225 exposure induces accumulation of hypophosphorylated retinoblastoma protein and increases expression of p27KIP1. An increase in Bax expression and concurrent decrease in Bcl-2 expression are observed when SCC cells are exposed to C225. Examination of C225 effects on radiation response in SCCs demonstrates enhancement in radiosensitivity and amplification of radiation-induced apoptosis. These effects are observed in both single-dose and fractionated radiation experiments. C225 represents a promising growth-inhibitory agent that can influence cellular proliferation, apoptosis, and radiosensitivity in SCCs of the head and neck.     

Stomatin‐like protein 2 inhibits cisplatin‐induced apoptosis through MEK/ERK signaling and the mitochondrial apoptosis pathway in cervical cancer cells

Stomatin‐like protein 2 (STOML2 or SLP‐2) is an oncogenic anti‐apoptotic protein that is upregulated in several types of cancer, including cervical cancer. However, the mechanisms responsible for the SLP‐2 anti‐apoptotic function remain poorly understood. Here, we show that siRNA‐mediated SLP‐2 suppression decreases growth of human cervical cancer HELA and SIHA cells, and increases cisplatin‐induced apoptosis through activation of MEK/ERK signaling and suppression of the mitochondrial pathway. The inhibition of the mitochondrial pathway is mediated by increasing the mitochondrial Ca²⁺ concentration and mitochondrial membrane potential, thereby downregulating p‐MEK and p‐ERK levels, upregulating the Bax/Bcl‐2 ratio, increasing cytochrome C release from mitochondria into the cytosol, and upregulating levels of cleaved‐caspase 9, cleaved‐caspase 3, and cleaved poly ADP‐ribose polymerase (PARP). Overexpression of SLP‐2 using adenovirus‐STOML2 has the opposite effect: it upregulates p‐MEK and p‐ERK and downregulates the Bax/Bcl‐2 ratio and levels of cleaved‐caspase 9 to caspase 9, cleaved‐caspase 3 to caspase 3, and cleaved‐PARP to PARP in cisplatin‐treated cells. These data show that SLP‐2 inhibits cisplatin‐induced apoptosis by activating the MEK/ERK signaling and inhibiting the mitochondrial apoptosis pathway in cervical cancer cells.     

Glycerol restores p53-dependent radiosensitivity of human head and neck cancer cells bearing mutant p53

Mutation or inactivation of p53 is known to be present in approximately 50% of human cancers. We propose here a novel strategy for overcoming this problem in mutant p53-targeting cancer therapies. We examined the restoration of radiation-induced p53-dependent apoptosis by a chemical chaperone (glycerol) in human head and neck cancer cells (SAS cells, showing wild-type p53 phenotype). SAS cells transfected with mutant p53 (SAS/m p53) showed radioresistance compared with SAS cells (SAS/ neo) transfected with neo vector as a control, but became radiosensitive when pre-treated with glycerol before X-ray irradiation. Apoptosis in the SAS/m p53 cells was induced by X-rays with glycerol pre-treatment, but not without glycerol pre-treatment, whereas apoptosis in the SAS/ neo cells was induced in both cases. Gel mobility-shift assays showed that after X-ray irradiation combined with glycerol pre-treatment, mp53 was able to bind to the sequence-specific region upstream of the bax gene regulating apoptosis. These results suggest that glycerol is effective in inducing a conformational change of p53 and restoring normal function to mp53, leading to enhanced radiosensitivity through the induction of apoptosis. This novel tool for enhancement of radiosensitivity in cancer cells bearing mp53 may be useful for p53-targeted radiotherapy.     

The immunohistochemical expression of DNA-PKCS and Ku (p70/p80) in head and neck cancers: relationships with radiosensitivity

PURPOSE: The DNA-PK complex is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. This study evaluated the relationship between the immunohistochemical expression of the individual components of DNA-PK and cellular radiosensitivity in head and neck cancers. METHODS AND MATERIALS: Biopsies from patients with previously untreated squamous cell carcinomas of the head and neck were assessed for inherent tumor radiosensitivity measured as the surviving fraction at 2 Gy (SF2) using a soft agar clonogenic assay. Paraffin-embedded tumor material from 64 successfully grown specimens was immunohistochemically stained for expression of DNA-PKcs and Ku (p70/p80). The same tumor material was previously analyzed for the immunohistochemical expression of p53. RESULTS: A significant correlation was found between the degree of expression of DNA-PKcs and Ku (p70/p80) (r = 0.55, p<0.001). There were no overall significant differences in the levels of expression of DNA-PKcs and Ku (p70/p80) in tumors from patients of either sex, different sites, histologies, and stages. No relationship was found between SF2 and the expression of either DNA-PKcs (r = 0.22, p = 0.081) or Ku (p70/p80) (r = 0.064, p = 0.62). Comparison with previous immunohistochemical characterization showed no significant correlations between the expression levels of p53 and either DNA-PKcs (r = 0.093, p = 0.46) or Ku (p70/p80) (r = -0.17, p = 0.17). CONCLUSIONS: This study suggests that determining the immunohistochemical expression of DNA-PK in head and neck cancers from multiple sites does not have a role as a predictive assay of tumor in vitro radiosensitivity.     

Downregulation of TMPRSS4 Enhances Triple-Negative Breast Cancer Cell Radiosensitivity Through Cell Cycle and Cell Apoptosis Process Impairment

Background: Radioresistance remains a challenge for cancer radiotherapy. The present study aims to investigate the role of TMPRSS4 in triple negative breast cancer (TNBC) cell radiosensitivity. Materials and Methods: After transfection of MDA-MD-468 triple negative breast cancer cells line by using the lentivirus vector, the effect of TMPRSS4 down-regulation on TNBC radiosensitivity was evaluated by using cloning assay and CCK-8 assay. The CCK-8 assay was also used for performing cell proliferation analysis. Western blot was carried out to detect the expression of certain proteins related to cell cycle pathways (cyclin D1), cell apoptosis pathways (Bax, Bcl2, and Caspase3), DNA damage and DNA damage repair (TRF2, Ku80 , ˠH2AX) . The cell cycle and cell apoptosis were also investigated using flow cytometer analysis. Results: TMPRSS4 expression was down-regulated in MDA-MB-468 cells which enhanced MDA-MB-468 cells radiosensitivity. TMPRSS4 silencing also improved IR induced cell proliferation ability reduction and promoted cell arrested at G2/M phase mediated by 6 Gy IR associated with cyclin D1 expression inhibition. Moreover, TMPRSS4 inhibition enhanced TNBC apoptosis induced by 6 Gy IR following by over-expression of (Bax, Caspase3) and down-regulation of Bcl2 as the pro-apoptotic and anti-apoptotic proteins, respectively. Otherwise, TMPRSS4 down-regulation increases  DNA damage induced by 6 Gy IR and delays DNA damage repair respectively illustrated by downregulation of TRF2 and permanent increase of Ku80 and ˠH2AX expression at 1 h and 10 h post-IR. Conclusion: Down-regulation of TMPRSS4 increases triple negative breast cancer cell radiosensitivity and the use of TMPRSS4 inhibitor can be encouraged for improving radiotherapy effectiveness in TNBC radioresistant patients.     

Microvessel density predicts the radiosensitivity of metastatic head and neck squamous cell carcinoma in cervical lymph nodes.

Cervical lymph node metastasis is the most common recurrence pattern of head and neck squamous cell carcinoma (HNSCC), and it is usually treated with radiation therapy and/or neck dissection. There has long been a desire for markers useful in predicting radiosensitivity to enable assignment of patients with recurrent head and neck cancer to clinical trials to improve their survival rates and quality of life. A total of 43 cases of HNSCC treated with whole or elective neck irradiation (total dose, 26-70 Gy; median, 60 Gy) for recurrent metastatic SCC in neck lymph nodes after neck dissection between 1992 and 1999 were the subject of this study. The relationship between radiosensitivity and clinicopathological and histopathological factors, including the Ki-67-labeling index for cell proliferation, p53 immunoreactivity and microvessel density (MVD), in surgical neck lymph node specimens were investigated by univariate and multivariate analysis. Of the 43 patients, 31 had recurrent tumors in neck lymph nodes after radiotherapy. Univariate analysis revealed significant associations between radiosensitivity and both high grade of keratinization (p=0.033) and low MVD (p=0.004), and marginally significant associations between radiosensitivity and grade of differentiation of the cancer in the lymph nodes (p=0.070). Multivariate analysis showed that only MVD had predictive value (p=0.016). Tumors with a high MVD possessed a significantly better neck control rate than tumors with a low MVD (p=0.004) by Kaplan-Meier analysis. MVD can be used as a good predictive marker for radiosensitivity of metastatic HNSCCs in cervical lymph nodes after neck dissection.     

Pilot study examining tumor expression of RAD51 and clinical outcomes in human head cancers

Radiation therapy and chemotherapy are commonly used treatments for head and neck cancer. RAD51 is a highly conserved DNA repair protein that serves a central function in the homologous recombination pathway. High levels of RAD51 protein expression have been reported in number of human cancer cell lines, and studies suggest that RAD51 overexpression can increase cellular resistance to radiation and some chemotherapeutic drugs. In this study, RAD51 protein levels were quantified by immunohistochemistry in tumor samples from twelve head and neck cancer patients who received identical treatment with induction chemotherapy (paclitaxel and carboplatinum) followed by radiation therapy given concurrently with additional chemotherapy (paclitaxel, fluorouracil, hydroxyurea). Patients with high RAD51 protein levels in their pre-treatment tumor biopsies demonstrated poorer cancer-specific survival rates than patients with lower RAD51 levels (33.3% vs. 88.9% at 2 years; p=0.025). In addition, within a subgroup of patients with normal tumor cell p53 expression, there was a non-significant trend toward better induction chemotherapy response rates observed in the tumors with lower RAD51 protein levels. These results suggest that tumor cell RAD51 expression levels may influence the outcome of patients with head and neck cancer treated with chemotherapy and radiotherapy.     

Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells

The aim of this study was to examine Bax, Bcl-2 and Bcl-XL proteins in human pancreatic cancer cell lines and to clarify the mechanism of radiation resistance. PANC-1 and AsPC-1 pancreatic cell lines were used, both having mutated p53. Radioresistant PANC-1/Rad cells and AsPC-1/Rad cells were obtained by repeated 5 Gy irradiation of PANC-1 cells and AsPC-1 cells, respectively. Radiation was found to inhibit the growth of PANC-1 cells and AsPC-1 cells. After exposure to radiation, detached cells were subjected to FITC-TUNEL staining to calcualte the ratio of apoptosis. TUNEL positive ratios increased dose-dependently in both cell lines. Western blotting showed that the basal level of the Bax/Bcl-2 ratio reflected the radiosensitivity of these cell lines, and Bax expression was obviously upregulated after irradiation in the presence of mutated p53, but Bcl-2 expression remained almost constant. Both PANC-1/Rad and AsPC-1/Rad cells had greater Bcl-XL expression than the parental cells, and the basal level of the Bax/Bcl-2 ratio was no longer predictive of radiosensitivity. Upregulated expression of Bax protein after irradiation was not related to induction of apoptosis in these cells, suggesting that overexpression of Bcl-XL and functional reconstruction of Bcl-2 family proteins are important factors in acquired radioresistance.     

miR-424-5p通过靶向抑制HMGA1表达提高宫颈癌放射敏感性

目的 探讨miR-424-5p对宫颈癌放射敏感性的影响及作用机制。方法 RT-qPCR检测miR-424-5p在宫颈癌组织和Hela细胞中表达;流式细胞术检测Hela细胞凋亡率;CCK-8检测Hela细胞增殖活性;蛋白印迹法检测Hela细胞中蛋白表达水平。结果 相较于正常组织和细胞,宫颈癌组织和Hela细胞中miR-424-5p表达量降低(1.03∶0.88,P<0.01;1.00∶0.75,P<0.001)。过表达miR-424-5p会抑制经放射处理后Hela细胞增殖活性(P<0.01),同时增加放射处理后Hela细胞凋亡率(24.82%∶49.94%,P<0.001)。过表达miR-424-5p会抑制HMGA1表达(1.01∶0.63,P<0.01),miR-424-5p会直接作用HMGA1进而影响宫颈癌放射敏感性。结论 miR-424-5p通过直接靶向HMGA1提高宫颈癌放射敏感性。     

Enhancement of radiosensitivity in head and neck cancer cells by ZD1839 ('IRESSA'), a selective epidermal growth factor receptor tyrosine kinase inhibitor

Overexpression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types, including head and neck squamous cell carcinomas (HNSCC). Recent laboratory experiments have demonstrated that high EGFR levels correlate with increased tumor resistance to radiation. This study investigated the relationship between EGFR expression levels and radiosensitivity in 5 HNSCC cell lines (HSC2, HSC3, HSC4, SCC25, and Ca9-22) and whether treatment with ZD1839 ('Iressa'), a selective EGFR-tyrosine kinase inhibitor (TKI), would improve tumor cell response to radiotherapy. ZD1839 suppressed the growth of HNSCC cell lines in a dose- and time-dependent manner. Radiosensitivity of these HNSCC cell lines, assessed by a clonogenic survival assay, differed greatly and the expression of EGFR varied. EGFR expression levels (EGFR numbers/cell) correlated with increased tumor resistance to radiation (f[x]= 4.54 X, R2 = 0.715; f[x]: EGFR numbers/cell, X: radiosensitivity; D10). Following exposure of the HNSCC cells to 1.0 microM ZD1839 and radiation (0-10 Gy), greater than additive growth inhibitory effects were observed. These results suggest that ZD1839 could enhance tumor radiosensitivity and inhibit tumor growth after radiation, indicating that this combination could have clinical potential in the treatment of patients with head and neck cancer.     

Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity.

Paclitaxel (PTX), a microtubule-active agent, blocks cell proliferation by inhibiting mitotic progression leading to mitotic and postmitotic arrest and cell death. Here we demonstrate for the first time that very low concentrations of PTX (3-6 nM) can completely inhibit cell proliferation without arresting cells at mitosis. At these low concentrations that are insufficient to inhibit mitotic progression, PTX induced both p53 and p21 causing G1 and G2 arrest in A549. In contrast, low PTX concentrations failed to induce G1 and G2 arrest in A549/E6 cells, that do not express p53. Furthermore, we observed that the levels of p53 and p21 induced by adriamycin and by low concentrations of PTX in A549 cells were comparable. This observation led us to conclude that low concentrations of PTX can induce p53 and p21 sufficiently to cause G1 and G2. Many other cell lines, including HCT116 cells, do not readily upregulate p53 in response to PTX, and therefore undergo exclusively mitotic and postmitotic arrest after PTX treatment. At low concentrations that do not cause mitotic arrest, PTX did not significantly inhibit proliferation of these cells. In HCT116 cells, loss of p53 (HCT/p53(-/-)) or p21 (HCT/p21(-/-)) affects both Bax and Bcl-2 expression. In cells lacking p53, levels of Bax and p21 were decreased. In cells lacking p21, levels of wt p53 were highly increased to compensate for the loss of p21. This in turn results in upregulation of Bax and downregulation of Bcl-2 resulting in an increase of the apoptotic Bax/Bcl2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. High levels of p53 and Bax/Bcl-2 ratio can also explain why loss of p21 is rarely found in human cancer.     

Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells.

Overexpression of wild-type p53 in cancer cells by adenovirus-mediated p53 gene transfer can result in the induction of apoptosis. To identify the potential mediators of this p53-induced apoptosis, we examined apoptotic protein levels in human lung cancer cells after Adp53 gene transfer. We observed up-regulation of Bax and Bak protein levels 18-36 h after transduction with Adp53 in H1299, H358, and H322 lung cancer cells. Contrary to expected observations, no changes in Bcl-2 and Bcl-X(L) protein levels were observed. Morphological cell changes and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining showed evidence of apoptosis in all cell lines 48 h after transduction with Adp53. These results indicate that the induction of apoptosis by adenovirus-mediated p53 transfer may be mediated by the induction of proapoptotic mechanisms rather than suppression of antiapoptotic mechanisms.     

Predictive value of P53, BCL-2, and BAX in advanced head and neck carcinoma

Because the apoptotic process appears to be involved in the response-to-treatment of chemotherapy and radiotherapy, we investigated the prognostic value of the expression of three apoptosis-associated genes (p53, Bax, and Bcl-2) in tumor biopsies from patients with locally advanced head and neck carcinoma. Using specific monoclonal antibodies, immunohistochemical staining for p53, Bax, and Bcl-2 was performed on tumor material from 43 patients before their scheduled adjuvant chemoradiotherapy. Results indicated that the response to treatment was 83.7% (36 of 43 patients). Bax staining was positive in 8 cases (19.5%), p53 in 19 (47.5%), and Bcl-2 in 4 patients (10.8%). There were no statistically significant correlations between any of the apoptosis genes assayed and the patients' response to treatment or to overall survival. In the univariate statistical analysis, response-to-treatment was the only significant variable (p = 0.013) predictive of survival rate. These results suggest that p53, Bax, and Bcl-2 expression are not significant predictive factors of response to induction treatment in locally advanced head and neck carcinoma and that their routine use as prognostic markers cannot be recommended.     

Synergic CSE1L/CAS, TNFR-1, and p53 apoptotic pathways in combined interferon-gamma/adriamycin-induced apoptosis of Hep G2 hepatoma cells

Many cancers are chemotherapy-resistant. Chemotherapy combined with immunotherapy offers a potential avenue for the treatment of chemotherapy-resistant cancers. In this study, we investigated the apoptotic pathways induced by combined interferon-gamma/adriamycin treatment in Hep G2 cells. Our data showed that Hep G2 cells treated with combined interferon-gamma/adriamycin enhanced cell apoptosis in comparison with that of cells treated with adriamycin. Interferon-y increased TNFR-1, CSE1L/CAS (cellular apoptosis susceptibility protein), Bax, and Bad levels. Adriamycin increased p53 and Bax, but not TNFR- 1 and CAS levels. Interferon-y did not increase p53 accumulation; nevertheless it enhanced adriamycin-induced p53 accumulation. Overexpression of IRF-1 augmented the combined interferon-gamma/adriamycin-induced p53 accumulation. Interferon-gamma co-treatment increased the stability of p53 protein induced by adriamycin. Our data suggest that TNF-gamma may greatly enhance the combined interferon-gamma/chemotherapeutic drug-induced apoptosis of cancers. Our findings also indicate that CAS, TN-FR-1, p53, Bax, and Bad may be the targets for the interferon-y-based chemo-immunotherapy of the chemotherapy-resistant cancers.     

Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines

Antitumor effects of the proapoptotic Bax gene have been evaluated in vitro and in vivo by a binary adenovirus system expressing the human Bax gene. Overexpression of the Bax gene in cultured cell lines from human lung carcinoma results in caspase activation, apoptosis induction, and cell growth suppression. Intratumoral injection of adenovirus vector expressing the Bar gene suppressed growth of human lung cancer xenografts established in nude mice. Histological examination of tumors from mice treated with the Bax gene demonstrated high levels of Bax expression and extensive apoptosis in tumors. In comparison with the treatment by an adenoviral vector expressing human p53, the Bax gene can effectively suppress tumor growth in both p53-sensitive and p53-resistant human lung carcinoma cell lines. Toxicity was not detected in liver and other systems in animals treated intralesionally with the Bax gene. Therefore, our results suggest that the Bar gene may be useful in cancer treatment.