Birgit Knerer

Betulinic acid: a new cytotoxic compound against malignant head and neck cancer cells.

A new compound, betulinic acid, has been found to be cytotoxic against a variety of tumor cells originating from the neural crest. Its efficacy against head and neck squamous cellular carcinoma cell lines has so far not been tested.

1,25(OH)2 vitamin D3 induces elevated expression of the cell cycle-regulating genes P21 and P27 in squamous carcinoma cell lines of the head and neck.

The biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], inhibits proliferation and induces differentiation for various malignant cells, including squamous cell carcinoma cell lines of the head and neck (SCCHN). These effects are due to an arrest of cells in the G0/G1 phase of the cell cycle and are predominantly mediated by the vitamin D receptor. To further explore the molecular mechanisms of the antiproliferative activity in SCCHN we studied the influence of 1,25(OH)2D3 on the expression of the G1 phase-regulating proteins cyclin D1, p21 and p27. Furthermore, as a direct target of G1 protein complexes, we investigated the phosphorylation status of the retinoblastoma protein (pRb). Synchronized cells of 2 SCCHN cell lines [JPPA (laryngeal carcinoma) and SCC 9 (tongue carcinoma)] and human immortalized keratinocytes (HaCaT) were cultured for 96 h in the presence or absence (ethanol as control) of 1,25(OH)2D3 (10(-7) M). At various time intervals the cell cycle status was detected by fluorescence-activated cell sorting (FACS) analysis and in parallel the expression of cell cycle-regulating proteins was determined at the protein and mRNA levels. In all cell lines tested 1,25(OH)2D3 caused an arrest of cells in the G0/G1 phase of the cell cycle and markedly induced the expression of the inhibitors p21 and p27. No influence was detectable on the expression of cyclin D1. Induction of p21 and p27 mRNA revealed transcriptional regulation by the vitamin D receptor. Simultaneously, hyperphosphorylated pRb was transformed to the hypophosphorylated form. Our results demonstrate that the biologically active form of vitamin D3 directly regulates the expression of p21 and p27, inducing a G0/G1 phase arrest: one mechanism by which 1,25(OH)2D3 controls cell proliferation inSCCHN.The biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], inhibits proliferation and induces differentiation for various malignant cells, including squamous cell carcinoma cell lines of the head and neck (SCCHN). These effects are due to an arrest of cells in the G0/G1 phase of the cell cycle and are predominantly mediated by the vitamin D receptor. To further explore the molecular mechanisms of the antiproliferative activity in SCCHN we studied the influence of 1,25(OH)2D3 on the expression of the G1 phase-regulating proteins cyclin D1, p21 and p27. Furthermore, as a direct target of G1 protein complexes, we investigated the phosphorylation status of the retinoblastoma protein (pRb). Synchronized cells of 2 SCCHN cell lines [JPPA (laryngeal carcinoma) and SCC 9 (tongue carcinoma)] and human immortalized keratinocytes (HaCaT) were cultured for 96 h in the presence or absence (ethanol as control) of 1,25(OH)2D3 (10-7 M). At various time intervals the cell cycle status was detected by fluorescence-activated cell sorting (FACS) analysis and in parallel the expression of cell cycle-regulating proteins was determined at the protein and mRNA levels. In all cell lines tested 1,25(OH)2D3 caused an arrest of cells in the G0/G1 phase of the cell cycle and markedly induced the expression of the inhibitors p21 and p27. No influence was detectable on the expression of cyclin D1. Induction of p21 and p27 mRNA revealed transcriptional regulation by the vitamin D receptor. Simultaneously, hyperphosphorylated pRb was transformed to the hypophosphorylated form. Our results demonstrate that the biologically active form of vitamin D3 directly regulates the expression of p21 and p27, inducing a G0/G1 phase arrest: one mechanism by which 1,25(OH)2D3 controls cell proliferation in SCCHN.

Magnetic cell separation for purification of human oral keratinocytes : an effective method for functional studies without prior cell subcultivation

Abstract In studying human oral keratinocytes, it would be very helpful to obtain a pure population of cells without prior in vitro expansion. An immunomagnetic separation technique, or magnetic cell separation (MACS), was modified for efficient purification of human oral keratinocytes. Subsequent to two-step enzymatic digestion, the cell suspension was labelled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells. In a second step a rat anti-mouse antibody conjugated with colloidal superparamagnetic particles was used. Labelled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. The unlabelled, unretained cells were further examined by flow cytometry analysis, enzyme-linked immunosorbent assay and polymerase chain reaction. After the MACS procedure, unretained cells showed a strong positivity for the lu-5 MoAb (as a marker for pan-cytokeratin) and were negative for anti-vimentin (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte-detecting antibodies, thus representing pure keratinocytes (> 98%). Purified keratinocytes maintained full viability (> 91%) and functional capacities. [3H]thymidine uptake and epidermal growth factor (EGF) receptor expression were unaltered when compared with the non-separated cell population. Furthermore, interleukin-1α was detected at the protein and RNA levels in keratinocytes immediately after MACS enrichment. Our findings show that MACS appears to be a useful tool for purification of oral keratinocytes and allows for further functional studies without prior subcultivation of cells.

Molecular analysis of p21 promoter activity isolated from squamous carcinoma cell lines of the head and neck under the influence of 1,25(OH)2 vitamin D3 and its analogs

Objective The biologically active 1,25(OH)2 vitamin D3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. For squamous carcinoma cell lines of the head and neck (SCCHN) we could show that this antiproliferative activity of 1,25(OH)2 vitamin D3 is due to induced expression of the cell-cycle inhibitory proteins p21 and p27, causing an arrest in the G0/G1 cell-cycle phase. Material and Methods In this work we investigated the effects of three vitamin D3 analogs, EB1089, MC1288 and CB1093, on proliferation behavior and cell-cycle status in a laryngeal carcinoma cell line (JPPA) as well as in control human immortalized keratinocytes (HaCaT). To study the molecular mechanism the functional activity of the promoter region of p21, a potential target gene of vitamin D3 transcriptional regulation, was investigated. For this reason a 2.7-kb fragment of the p21 promoter was isolated by polymerase chain reaction from HaCaT, JPPA and SCC9 (tongue carcinoma) cells and directionally cloned into an enhanced green fluorescence protein (EGFP) reporter gene vector system. A construct was used to stably transfect HaCaT cells and to monitor the expression of the EGFP gene by confocal microscopy. Results Analysis of proliferation and cell-cycle status revealed decreased growth rates and G0/G1I cell-cycle arrest in cells treated with 1,25(OH)2 vitamin D3 and its analogs The EGFP reporter gene-transfected cells showed distinct fluorescence under the influence of 1,25(OH)2 vitamin D3 and its analogs compared to control cells. Conclusions These results demonstrate that the cell-cycle inhibitor protein p21 is a direct target gene of biologically active 1,25(OH)2 vitamin D3, inducing G0/G1 cell-cycle arrest. The ability of vitamin D analogs to act via the same molecular mechanism as the natural hormone but with less hypercalcemic activity may have therapeutic implications for patients with SCCHN malignancy.

Treatment of a local recurrence of a carcinoid tumor of the middle ear by extended subtotal petrosectomy

Abstract A recurrence of a primary carcinoid tumor of the middle ear 15 years after radical tympanomastoidectomy is reported. An extended subtotal petrosectomy using a craniocervical approach with temporary infracondylar mandibulotomy was performed, since imaging studies demonstrated an extensive tumor with a close relationship to the tegmen tympani, facial nerve, and ascending and horizontal portions of the carotid canal. The tumor was metabolically inactive. Histopathological examination showed a solid, trabecular tumor that was positive for pancytokeratin Lu5, neuron-specific enolase, pancreatic intestinal polypeptide and glucagon. Neuroendocrine-granules were demonstrable under electron microscopy. This case is reported to show that primary middle-ear carcinoid tumors can recur years after radical tympanomastoidectomy.

Antiproliferative effects of the biologically active metabolite of vitamin D3 (1,25 [OH]2 D3) on head and neck squamous cell carcinoma cell lines

In addition to a role in calcium and phosphate homeostasis other vitamin D receptor (VDR) mediated effects have been discovered during the past few years for the biologically active metabolite of vitamin D, 1,25(OH)2D3. An antiproliferative, differentiation-inducing effect on nonmalignant and neoplastic cells of different origin has now been described. We examined the influence of 1,25(OH)2D3 on human squamous cell carcinomas of the head and neck (SCCHN). A differentiated (JP-PA) and undifferentiated (LF-FR) SCCHN line was studied with respect to proliferative capacity (using [3H]-thymidine uptake and cell number) and cell cycle distribution as determined by flow cytometry (FACS). Both cell lines were positive for VDR, which was found to be increased after the addition of 10−7 M1,25(OH)2D3, as shown by FACS analyses. The administration of 1,25(OH)2D3 at a concentration between 10−7 M and10−10 M caused a dose-dependent moderate growth inhibition, as reflected by down-regulation of DNA synthesis (reduced [3H]-thymidine uptake) and a decrease in cell numbers. The JP-PA cell line showed a significant growth reduction for both concentrations tested, whereas for LF-FR a significant inhibition was detected only for 10−7 M. The addition of 10−7 M 1,25(OH)2D3 caused a blockade in the transition of cells from G1 to S phase in both cell lines, with a significant accumulation of cells in the G0/G1 phase. Our results demonstrate a receptor-mediated, dose-dependent inhibition of neoplastic growth by 1,25(OH)2D3 in human SCCHN lines.

Non-steroidal anti-inflammatory drugs induce apoptosis in head and neck cancer cell lines.

Non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis in colorectal cancer due to the induction of apoptosis. Disturbances of cellular pathways ultimately leading to apoptosis may contribute to the process of neoplastic transformation and immortalization. In this study we wanted to determine the influence of different NSAIDs (indomethacin, ibuprofen and sodium salicylate) and hydrocortisone on Bcl-2 expression and the apoptotic behavior of head and neck tumor cell lines and normal oral keratinocytes. Bcl-2 expression was determined by monoclonal antibody staining and fluorescence-activated cell-sorting measurement. Apoptotic cells were visualized with a epifluorescence microscope after staining with CytoDeath M30 antibody. Indomethacin (1 mM) and ibuprofen (1 mM) significantly reduced Bcl-2 expression in the cancer cell lines tested and might be thought responsible for the observed increase in apoptosis. At all concentrations tested the influence of sodium salicylate and hydrocortisone on Bcl-2 expression was not significant. In contrast, the NSAIDs tested had only a minor influence on normal oral keratinocytes. Our results demonstrate a significant reduction in growth and an increase in apoptosis, possibly due to a reduction in Bcl-2 expression. after exposure to indomethacin and ibuprofen in the head and neck cancer cell lines tested.Non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis in colorectal cancer due to the induction of apoptosis. Disturbances of cellular pathways ultimately leading to apoptosis may contribute to the process of neoplastic transformation and immortalization. In this study we wanted to determine the influence of different NSAIDs (indomethacin, ibuprofen and sodium salicylate) and hydrocortisone on Bcl-2 expression and the apoptotic behavior of head and neck tumor cell lines and normal oral keratinocytes. Bcl-2 expression was determined by monoclonal antibody staining and fluorescence-activated cell-sorting measurement. Apoptotic cells were visualized with a epifluorescence microscope after staining with CytoDeath M30 antibody. Indomethacin (1 mM) and ibuprofen (1 mM) significantly reduced Bcl-2 expression in the cancer cell lines tested and might be thought responsible for the observed increase in apoptosis. At all concentrations tested the influence of sodium salicylate and hydrocortisone on Bcl-2 expression was not significant. In contrast, the NSAIDs tested had only a minor influence on normal oral keratinocytes. Our results demonstrate a significant reduction in growth and an increase in apoptosis, possibly due to a reduction in Bcl-2 expression, after exposure to indomethacin and ibuprofen in the head and neck cancer cell lines tested.

IL-1 and TNF-α but no IL-2 Expression is Found in Squamous Cell Carcinomas of the Head and Neck by RT-PCR

We studied the expression of IL-1 (Interleukin 1), IL-2, IL-4, IL-10, GM-CSF (granulocyte/macrophage colony stimulating factor), TNF-alpha (tumor necrosis factor alpha) and IFN-gamma (Interferon gamma) in tumor specimens and adjacent mucosa from 12 patients with squamous cell carcinoma of the head and neck (SCCHN) by reverse-transcribed polymerase chain reaction (RT-PCR). No IL-2 and IL-4 expression was detected in any of the tumor specimens. High level expression of IL-1 and TNF-alpha was found in all 12 samples analyzed. Expression of GM-CSF was detected in 7 (58%) of 12 tumors, IL-10 was found in 3 (25%) and IFN-gamma in 6 (50%) of the samples. No qualitative differences in the cyotokine expression pattern were found between normal and tumor tissue. Our data indicate that there is a lack of cytokines typical for a specific, T-cell mediated immune response in SCCHN. A number of cytokines which are required for the initial steps of immune response however are produced. These results might have implications for future immuno-chemotherapeutical strategies.

Mitoxantrone and cisplatin in recurrent and/or metastatic salivary gland malignancies.

A phase II study was performed to assess the safety and efficacy of mitoxantrone and cisplatin in locally recurrent and/or metastatic carcinomas of the salivary glands. Between May 1997 and March 2001, a total of 14 patients were entered on this trial. All of them had previously undergone radical resection and 10 were subsequently treated with adjuvant radiation therapy with (n =3) or without (n =7) concomitant chemotherapy. Therapy according to the study protocol consisted of mitoxantrone given as i.v. bolus on day 1 at a dose of 12 mg/m2 and cisplatin given as 90-min infusion at a dose of 30 mg/m2 on days 1–3. We observed two partial responses (14.3%) and stabilization of disease in nine patients (64.3%); progression during therapy was noted in only three cases (21.4%). The median time to progression was 15 months (range 2–36) and the median survival time was 27 months (range 4–54). Myelosuppression was commonly observed. Leukocytopenia occurred in all patients, and was grade 3 or 4 in three (21%) and four (29%) patients. WHO grade 3 thrombocytopenia and anemia was seen in three (21%) and four (29%) patients, respectively. Non-hematologic toxicity was in general mild to moderate except for two cases (14%) of grade 3 nausea and vomiting; overall incidence rates were nausea and vomiting (n =14), stomatitis (n =6), diarrhea (n =3), alopecia (n =11), infection (n =7), increase of serum creatinine (n =3), and peripheral neuropathy (n =3). The combination of mitoxantrone and cisplatin seems to be an active and fairly well-tolerated regimen for the treatment of advanced salivary gland cancers. According to the observed high rate of abrogating progressive disease for a long duration, and the resulting promising progression-free and overall survival time, further investigation seems warranted.

Cytokine Expression of Human Oral Keratinocytes

The present study aimed to examine the cytokine expression pattern of human oral mucosa-derived keratinocytes at the protein and RNA level. The mRNA expression was measured by a RT-PCR method and the protein production was determined by an ELISA technique. In freshly isolated oral keratinocytes, IL-1α (interleukin 1α), IL-6, IL-8, transforming growth factor β, tumor necrosis factor α and basic fibroblast growth factor were detectable at the protein and mRNA level, whereas platelet-derived growth factor, acidic fibroblast growth factor and transforming growth factor α were found only at the mRNA level. There were no detectable signals of IL-2 and IL-4. The cytokine production at the protein level was independent of prior stimulation with phorbol myristate acetate. Our results show that human oral keratinocytes – under nonpathological conditions – produce a cytokine pattern quite similar to that of epidermal keratinocytes, which may have implications for further functional studies.