Apoptotic effect in combination of Cyclosporin A and Taxol on oral squamous cell carcinoma cell line through the PI-3 kinase/Akt1 Pathway

Oral cancer take up 2-6% of all carcinomas and squamous cell carcinoma, which is the most common type in oral cancer, has a poor prognosis due to its high metastasis and recurrence rates. In treating oral cancer, chemotherapy to the primary, metastasized and recurrent lesion is a very important and useful treatment, even though its widespread usage is limited due to high general toxicity and local toxicity to other organs. Taxol, a microtubule stabilizing agent, is an anticancer drug that induces cell apoptosis by inhibiting depolymerization of microtubules in between the metaphase and anaphase of the cell mitosis. Recently, its effectiveness and mechanism on various tumor has been reported. However, not much research has been done on the application of Taxol to oral squamous cell carcinoma. Cyclosporin A, which is an immunosuppressant, is being used on cancers and when co-administered with Taxol, effectiveness of Taxol is enhanced by inhibition of Taxol induced multidrug resistance. In this study, Cyclosporin A with different concentration of Taxol was co-administered to HN22, the oral squamous cell carcinomacell line. To observe the cell apoptosis and the mechanisms that take part in this process, mortality evaluation of tumor cell using wortmannin, c-DNA microarray, RT-PCR analysis, cytometry analysis and western blotting were used, and based upon the observation on the effect and mechanism of the agent, the following results were obtained: 1. The HN22 cell line viability was lowest when 100micrometer of Wortmannin and 5microgram/ml of Taxol were co-administered, showing that Taxol participates in P13K-AKT1 pathway. 2. In c-DNA microarray, where 1microgram/ml of cyclosporine A and 3mg/ml of Taxol were co-administered, no up regulation of AKT1, PTEN and BAD c-DNA that participate in cell apoptosis was observed. 3. When 1microgram/ml of Cyclosporin A was applied alone to HN22 cell line,no difference was found in AKT1, PTEN and BAD mRNA expression. 4. Increased AKT1, mRNA expression was observed when 3microgram/ml of Taxol was applied alone to HN22 cell line. 5. When 1microgram/ml of Cyclosporin A and Taxol (3microgram/ml and 5microgram/ml) were co-administered to HN22 cell line, PTEN mRNA expression increased, whereas AKT1 and BAD mRNA decreased. 6. As a result of cytometry analysis, in the group of Cyclosporin A(1microgram/ml) and Taxol(3microgram/ml) co-administration, increased Annxin V was observed, which shows that apoptosis occurred by deformation of plasma membrane. However, no significant difference was observed with varying concentration. 7. In western blot analysis, no caspase 3 was observed in the group of Cyclosporin A(1microgram/ml) and Taxol(3microgram/ml) co-administration. From the results of this study, it can be concluded that synergistic effect can be observed in combination therapy of Taxol and Cyclosporin A on oral squamous cell carcinoma cell line, where decreased activity of the cell line was observed. This resulted in decreased AKT1 and BAD mRNA and increased PTEN mRNA expression and when wortmannin and Taxol were co-administered, the viability decreased which confirms that Taxol decreases the viability of tumor cell line. Hence, when Taxol and cyclosporine A are co-administered, it can be assumed that cell apoptosis occurs through AKt1 pathway.

Expression of Anaphase Promoting Complex in Surgically Resected Squamous Cell Carcinoma and Adenocarcinoma of the Lung

BACKGROUND: The anaphase promoting complex (APC) promotes the degradation of mitotic cyclins as well as other substrates involved in sister chromatid adhesion. This study was carried out to examine the relationship between the APC expression and the clinicopathological variables, in an attempt to determine the role of the APC in the proliferation of lung cancer and to evaluate the possibility of an aberrant APC function in surgically resected squamous cell carcinomas and adenocarcinomas of the lung. METHODS: Immunohistochemical staining was performed for APC, Ki-67, cyclin B1, Cdc2, MMP-2 and VEGF in 55 cases of squamous cell carcinoma and 34 cases of adenocarcinoma of the lung, using the avidin-biotin-peroxidase method. RESULTS: The immunohistochemical stains for APC revealed a positive reaction in 49 cases (55.1%). The APC expression level was higher in the cyclin B1-positive group (p= 0.01), the Cdc2-positive group (p=0.001), the MMP-2-positive group (p=0.03), the group with lymph node metastasis (61.4% vs 48.9%), and the group with stage II/III cancer (60.7%) compared with those with stage I (42.9%). CONCLUSIONS: The APC may have an aberrant function, such as a change in its role in controlling the cell cycle, and might be associated with the invasiveness and proliferation of tumor cells.

Sec13 induces genomic instability in U2OS cells

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.

Expression of Anaphase Promoting Complex (APC) and APC Regulatory Proteins in Invasive Ductal Carcinoma Associated with Paget's Disease

BACKGROUND: Oncogene expression in Paget's disease of the breast is not well known. To characterize invasive ductal carcinoma associated with Paget's disease, we studied expression of anaphase promoting complex (APC) with its regulatory proteins. METHODS: Immunohistochemical stainings were done with 10 cases of invasive ductal carcinoma associated with Paget's disease for APC, pituitary tumor transforming gene (PTTG), cyclin B1, p53, cyclin D1, and c-erbB-2. The expressions of these markers in Paget's disease were compared with those in the associated with carcinoma. RESULTS: APC, PTTG, cyclin B1, and c-erbB-2 were positive in all of the cases with both Paget's disease and underlying carcinoma. p53 was expressed in Paget's disease of 6 cases (60%) and in carcinoma of 7 cases (70%). Cyclin D1 was positive in Paget's disease of 8 cases (80%) and in carcinoma of 9 cases (90%). CONCLUSIONS: Breast carcinomas with Paget's disease seem to be distinguished by the high expression of APC, cyclin B1, PTTG, c-erbB2, and cyclin D1 in contrast to breast cancers without Paget's disease. Furthermore, the similar expression patterns of APC and APC regulatory proteins in both Paget's disease and underlying breast cancer support the epidermotropic theory as its pathogenetic mechanism.

Contribution to the validation of the anaphase-telophase test: aneugenic and clastogenic effects of cadmium sulfate, potassium dichromate and nickel chloride in chinese hamster ovary cells

As evidências de que a aneuploidia durante a mitose pode ser um fator na etiologia de malignidades somáticas estäo cada vez mais fortes. A análise de alteraçöes em anáfase-telófase da mitose é um teste útil para a avaliaçäo da capacidade aneuploidogênica e clastogênica de substâncias químicas. Vários metais têm sido identificados como carcinogênicos para o homem e para animais. Contudo, os mecanismos de açäo permanecem obscuros. No presente estudo, a capacidade aneugênica e clastogênica do sulfato de cádmio, do dicromato de potássio e do cloreto de níquel foi analisada usando o teste anáfase-telófase. Células do ovário do hamster chinês cultivadas por dois ciclos foram tratadas com o composto desejado por 8 horas antes da colheita das células. Foram quantificadas as freqüências de células com pontes de cromatina, lagging cromossomos e lagging fragmentos cromossômicos. O índice mitótico foi determinado pela contagem do número de células em mitose por 1000 células em cada lamínula e foi expresso como uma porcentagem do número de placas mitóticas. A análise estatística foi feita usando o método "G". Análises de correlaçäo e de regressäo foram realizadas para avaliar as variaçöes do índice mitótico. O crômio e o cádmio foram clastogênicos e aneugênicos e aumentaram as freqüências dos três tipos de aberraçöes avaliadas; o níquel teve apenas atividade aneugênica porque ele aumentou a freqüência de lagging cromossomos. Estes resultados indicam que o teste anáfase-telófase é sensível o suficiente para detectar as relaçöes dependentes da dose que podem distinguir as atividades clastogênicas e/ou aneugênicas e que os resultados obtidos usando o teste anáfase-telófase foram semelhantes aos obtidos pela contagem cromossômica.

In vitro induction of abnormal anaphases by contaminating atmospheric dust from the city of Mexicali, Baja California, Mexico

Mexicali dust (MD) is a mixture of particles of potassium aluminum silicates (98 percent) and sodium dioxide (2 percent) that induces pulmonary damage under experimental conditions, and is capable of inducing in vitro chromosomal alterations in exposed lymphocytes. It has been proposed as an atmospheric contaminant with pathogenic potential. among the chromosomal alterations observed, numeric alterations were predominant. The present study was designed to evaluate the capacity of MD to induce anaphasic changes in the Balb c 3T3 cell line. Chrysotile asbestos was used as a positive control. MD was found to induce abnormal anaphases, and the percentage of abnormalities increased as the dose increased (27.41 percent with 20 mg/mL, 29.60 percent with 40 mg/mL and 37.10 percent with 80 mg/mL). Multipolar anaphases constituted the most frequent altertion (69.1 - 78.8 percent), followed by lagging chromosomes (18.2 -29.5 percent) and anaphasic bridges (1.51 - 5.9 percent). The anaphasic alterations induced by MD showed differences in comparison to those observed with asbestos, especially for anaphasic bridges (10.4 percent vs. 1.51 percent, p<0.05). The capacity of MD to induce alterations resported in the process of chromosomal disjunction could explain the numeric aberrations reported previously by the authors of this paper. Therefore, these data support that MD could act as a clastogenic agent

C-anaphases (Common Variant) are unrelated to colchicine and phytohemagglutinin

We wvaluated the effect of different concentrations of colchicine (0.0, 0.1, 0.3, 0.5 µg/ml) and phytohemagglutinin (PHA) (0.10, 0.15, 0.20 µg/ml) on the rate of C-anaphases in lymphocyte cultures from five healthy individuals with the common variant of C-anaphases. For each of the 12 possible combinations, two subjects were randomly tested. The frequency of these variant figures was <3 percent; a single culture (out of six with the colchicine concentration of 0.1 µg/ml) lacked C-anaphases. Multiple variance and Student's t tests revealed, as the only significant difference, a decrease with the colchicine concnetration of 0.3 µg/ml compared with the cultures without colchicie (p<0.05), which exhibited the greatest ratio of C-anaphases. The PHA had no influence on the frequency of C-anaphases. We conclude that the common variant of C-anaphases is unrelated to the colchicine and PHA concentrations tested; moreover, our data confirm the occurrence of such a mitotic variant in colchicine-free cultures

Inducción de anafases anormales por polvos inorgánicos en cultivo de celulas BALBC/3T3 / Induction of abnormal anaphases by inorganic dusts in culture of BALBC/3T3 cells

Los polvos inorgánicos como el asbesto son capaces de inducir alteraciones cromosómicas in vitro. Estas alteraciones se han comprobado empleando fibras de longitud mayores de 5 µm. En el presente estudio se valoraron las anafases anormales inducidas por fibras de asbesto crisótilo y por hierro carbonilo con un tamaño menor de 5.0 µm. Se sembraron 10 células BALBC/3T3 en medio RPMI-1640 y se expusieron a las siguientes dosis de cada polvo: 0, 5, 10, 20, 40, 80 µg/ml durante 12 horas. Las células se fijaron y tiñeron con safranina-O para valorar las anafases anormales inducidas. Se observó que el asbesto crisótilo fue capaz de disminuir las anafases totales conforme de dosis de éste se elevó (de 36 por ciento a 8 por ciento), y el número máximo de anafases anormales fue de 5.6 por ciento con 40 µg/ml. A esta dosis, las anormalidades más comunes fueron los puentes anafásicos (4.3 por ciento) y las anafases multipolares (1.6 por ciento). El hierro carbolino no indujo disminución de las anafases y las anormales inducidas no fueron estadísticamente diferentes de las observadas en cultivos control. De nuestros resultados podemos concluir que las fibras de asbesto crisótilo menores de 5 µm de longitud inducen daño cromosómico directo e indirecto en cultivo celular

Mutagenic bioassay of certain pharmacological drugs: II. Mebendazole (MBZ)

El Mebendazole (MBZ), o 5-benzoyl- 1H-benzimidazole-2- il ácido metil éster carbámico fue estudiado con tres ensayos de corto plazo. Se probó su capacidad para inducir micronúcleos "in vivo" en eritrocitos policromáticos de médula ósea de ratones CFW. Se analizaron 3 dosis, 25, 50 y 100 mg/kg de peso corporal inyectados intraperitonealmente. Las 3 dieron incremento significativo (p < 0.01). Las pruebas "in vitro" se realizaron en células CHO. Se analizó la capacidad para inducir aberraciones cromosómicas y figuras anormales por prueba de anafase-telofase. Se detectó una frecuencia aumentada en la formación de dicéntricos, gaps, C-mitosis, puentes y cromosomas rezagados. La acción genotóxica observada puede asociarse con la estructura química de un æester carbámico derivado que sugeriría un papel de mutágeno directo

Post-metaphase mitotic events in cells treated with dinitrophenol.

The sequence of post-metaphase mitotic events, such as anaphase movement A and B, chromosome decondensation, nuclear envelope reformation and cytokinesis, has been studied in 2,4-initrophenol (DNP)-treated HeLa cells. The effects of DNP were found to be dose dependent and at concentrations higher than 3 mM, both anaphase A and B movements were totally and nearly instantaneously arrested. It could be shown that cytokinesis did not depend on the completion of anaphase movements. This was also true for nuclear envelope reformation which could take place even around condensed chromosomes arrested in anaphase. The post-metaphase mitotic events do not follow a strict causal sequence, but they can be dissociated from each other in anaphase-arrested cells.