Same number of untreated cells was used as control. All the inserts were put in

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 Same number of untreated cells was used as control. All the inserts were put in Empty Same number of untreated cells was used as control. All the inserts were put in

Post  wangqian on Tue Jun 10, 2014 4:39 am

After the pre culture, the six different cell culture groups were subjected to a conven tional abt263 matrigel assay. Only HUH REISO cells, pre cultured by agarose overlay, showed enough plas ticity to form endothelial like tubes within 4h. After 24h, the network was fully trained in this group, whereas HUH wt and HUH PAS cells showed no striking tube forma tion. Moreover, tube formation was not detectable in all three tumor cell groups pre cultured under conventional conditions. Quantification of tube formation in matrigel was performed via software based analysis. Comparison of HUH wt, HUH PAS, and HUH REISO revealed a far higher number of branching points and tubes, a very extended length of skeleton, and a decreased amount of confluent areas without tube formation for HUH REISO cells.

Furthermore, expression levels of endothelial markers of agarose overlay HUH REISO cells, showing posi tive tube formation, were compared to the corresponding standard culture HUH REISO. Expression levels were determined after the matrigel assay. In agarose overlay HUH REISO cells, ICAM 2. as well as CD31 PECAM 1 were significantly upregulated. Expression Adriamycin Topoisomerase 阻害剤 of the other endothe lial markers was not detectable in HUH REISO under any culture condition. Discussion In the present study, acquired in vivo chemoresistance against metronomic cyclophosphamide treatment was studied in a human hepatocellular carcinoma HUH7 xenograft mouse model. During treatment, a two phase development of tumor progression was observable In the beginning of treatment, tumor pro gression was significantly decreased, indicated by con stant tumor volume for about 75 days.

In the following, second phase, tumor volume increased with a tumor volume doubling time of 3. 5 days, des pite ongoing ABT199 therapeutic intervention. Viable tumor cells were extracted from resistant tumors, whereas control cells were obtained from in vivo passaging HUH7 tumor cells without CPA treatment. Subsequently, HUH PAS and HUH REISO were characterized and identified in terms of cell morph ology and representative human epidermal growth fac tor expression for their human origin. Interestingly, in vivo chemoresistant HUH REISO did not manifest their drug resistant phenotype in a two dimensional monolayer culture in presence of in situ ac tivated CPA.

In addition, significant changes in macroscopic appearance and tumor tissue organi zation of chemoresistant tumors indicate resistance mechanisms, which were only gainful in the in vivo situ ation. However, an endogenous imprinted component for in vivo chemoresistance was obvious, as the chemo resistant phenotype of isolated tumor cells was immedi ately manifested again after reimplantation and reapplied chemotherapy. In the reimplantation experiment, che moresistance was manifested lacking the response phase. In contrast, HUH PAS, which were only adjusted to the in vivo environment but not to the treatment, remained sensitive in a response phase. The qRT PCR assay on in vivo samples revealed no significant differ ence in basal expression of the detoxification enzyme ALDH 1, which converts aldophosphamide into carboxyphosphamide.


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