di dalam hatiku hanya satu nama yang ada di tulus hati ku ingini

kesetiaan yang indah takkan tertandingi hanyalah dirimu satu peri cintaku

benteng begitu tinggi sulit untuk ku gapai..

akununutk kamu, kamu untuk aku

namun semua apa mungkin iman kita yang berbeda,,

tuhan memang satu, kita yang tak sama

haruskah aku lantas pergi meski cinta takkan pernah pergi

aku tak akan pernah lupa dengan dirinya yang telah membuat aku sakit hati :@

sandiwara cinta

jujurlah sayang, aku tak mengapa

biar semua, jelas dan berbeda

jika nanti aku yang harus pergi

kuterima walau sakit hati...

buat seseorang yang kubenci.....!!!!

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1. Dudeck O, Ricke J. Advances in regional chemotherapy of the liver. Expert Opin Drug Deliv. 2011;8:1057-1069

2. Jemal A, Bray F, Center MM. et al. Global cancer statistics. CA Cancer J Clin. 2011;61:69-90

3. Zhou BB, Zhang H, Damelin M. et al. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov. 2009;8:806-823

4. Al-Haj

Because of different experiment methods and cell types in every research group, the confusion results have been found in HCC. Recently, CD90 and CD13 have been used to identify HCC TICs, while CD44 and CD24 are mainly used as breast or pancreatic TICs markers [4, 10, 16, 20]. Using flow cytometry assay, we found the low expression of CD90, CD13 or CD44, and high expression of CD24 in HCC Bel7404, Bel7402, Huh7, SMMC7721 and HepG2 cells (data not shown). Otherwise, EpCAM or CD133 as an efficient marker of TICs has been proved by many researches in HCC, especially in Huh7 cells. Thus, we chose EpCAM and CD133 as the biomarkers of TICs.

In spite of single marker, two markers are combined to identify and isolate TICs precisely. CD133⁺CD44⁺ phenotype in SMMC7721, MHCC-LM3, and MHCC-97L cells [17], CD133⁺ALDH⁺ phenotype in PLC8024 cells [22], and CD45^-CD90⁺ phenotype isolated from human tumor specimens and blood samples [16] are significantly more tumorigenic both in vitro and in vivo. Using other combination of surface markers, sorted Huh7 cells can not generate tumors in NOD/SCID mice [17]. However, we found that only 500 CD133⁺EpCAM⁺ cells formed tumors in NOD/SCID mice, rather than single marker positive cells. It was notable that CD133⁺EpCAM⁺ cells also possessed more characteristics of TICs rather than CD133⁺EpCAM^-, CD133^-EpCAM⁺ or CD133^-EpCAM^- cells in vitro. It suggests that CD133⁺EpCAM⁺ are the effective combination of TICs surface markers in Huh7 cells. Furthermore, recent clinic researches have shown that CD133 or EpCAM, associated with recurrence, alone may not be sufficient to serve as prognostic parameter of HCC [24, 41]. It is worthwhile for further investigation of CD133 and EpCAM in clinical significance.

CD133⁺EpCAM⁺ cells in SP cells were much more than in non-SP cells. Enriched SP cells were mainly composed of CD133⁺EpCAM⁺ cells. These data suggested that cells, co-expressing CD133 and EpCAM, possessed the characteristics of SP cells. Recently, SP cells are proved to be associated with drug-resistant [30]. Consistent with these results, our study showed that CD133⁺EpCAM⁺ cells possessed drug-resistant characteristic.

Stem cell-related genes Nanog, Sox2 and Oct4 are important for proliferation, self-renewal, and differentiation of stem cells [42-43]. Stem cell-related genes have been reported in TICs recently [13, 18]. In this study, stem cell-related genes were also up-regulated in CD133⁺EpCAM⁺ cells. These might be the causes for the enhancement of self-renewal and differentiation in CD133⁺EpCAM⁺ cells, but need for further investigation.

 Figure 6 

CD133⁺EpCAM⁺ cells possessed other characteristics of TICs. A. Various phenotypes were collected, and then the expression of Nanog, Sox2 and Oct4 were measured by real-time PCR. Data were from triple independent experiments. B. Cell spheres were imaged by microscope (x200 fields) after cultured in modified medium for 7 days. Data were shown as mean ± SD of three independent experiments. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, #P<0.05 to CD133⁺EpCAM^-.

(Click on the image to enlarge.)

 Figure 7 

Raised tumorigenicity of CD133⁺EpCAM⁺ cells was detected in NOD/SCID mice. A. The indicated numbers of various phenotypes were injected subcutaneously in NOD/SCID mice for 90 days. The incidence of tumors was examined bi-weekly. B. 1,000 CD133⁺EpCAM⁺ or CD133^-EpCAM^- cells were injected in the indicated place for 90 days. C. The tumors were stained with hematoxylin-eosin. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, #P<0.05 to CD133⁺EpCAM^-.

(Click on the image to enlarge.)

To observe the effects of CD133 or EpCAM, CD133⁺EpCAM^- cells were compared with CD133^-EpCAM⁺ cells in Huh7 cells. Exception of high expression of stem-related genes in CD133⁺EpCAM^-, there was no significant difference between two markers in vitro. In vivo, CD133⁺EpCAM^- cells had been found more tumorigenic potential than CD133^-EpCAM⁺ cells. It suggested that CD133 had more relative characteristics of TICs than EpCAM. Moreover, EpCAM has been proved to be a direct transcriptional target in the Wnt/β-catenin signaling pathway [44]. CD133 has been decreased through the blockade of Notch pathway [45]. It is worthwhile for further study of these signaling pathways on CD133 and EpCAM in Huh7 cells.

Although the high expression of EpCAM in Bel7402 cells, there was no obvious difference between EpCAM⁺ and EpCAM^- cells in vitro, including colony and sphere formation ability (data not shown). Meanwhile the expression of CD133 and EpCAM were low in HepG2 cells, which could not form subcutaneous tumor in ATCC. It was hypothesized that there might be another surface marker in Bel7402 or HepG2 cells with different cellular origin.

After flow cytometry sorting, CD133⁺EpCAM⁺ cells differentiated into the other phenotypes in medium with FBS, which was confirmed by other group [18]. Recently, it has been reported that spheroid cells expressed TICs characteristics [38]. Taken together, the combination of multi-marker sorting and the spheres formation might be the better strategy to maintain the TICs characteristics in a restricted period of time. It will be more suitable for studying basic biology of TICs and screening new drug targets.

In conclusion, our data suggest that CD133⁺EpCAM⁺ phenotype precisely represented the characteristics of TICs in Huh7 cells, including self-renewal, differentiation, resistance, tumorigenicity. It may promote the basic research on TICs and drug-screening.

Abbreviati